Detection of transmembrane potentials by optical methods

ABSTRACT

Methods and compositions are provided for determining the potential of a membrane. In one aspect, the method comprises:  
     (a) introducing a first reagent comprising a hydrophobic fluorescent ion capable of redistributing from a first face of the membrane to a second face of the membrane in response to changes in the potential of the membrane, as described by the Nernst equation,  
     (b) introducing a second reagent which labels the first face or the second face of the membrane, which second reagent comprises a chromophore capable of undergoing energy transfer by either (i) donating excited state energy to the fluorescent ion, or (ii) accepting excited state energy from the fluorescent ion,  
     (c) exposing the membrane to radiation;  
     (d) measuring energy transfer between the fluorescent ion and the second reagent, and  
     (e) relating the energy transfer to the membrane potential.  
     Energy transfer is typically measured by fluorescence resonance energy transfer. In some embodiments the first and second reagents are bound together by a suitable linker.  
     In one aspect the method is used to identify compounds which modulate membrane potentials in biological membranes.

[0001] This invention was made with Government support under Grant No.R01 NS27177-07, awarded by the National Institutes of Health. TheGovernment has certain rights in this invention.

BACKGROUND OF THE INVENTION

[0002] The present invention relates generally to the detection andmeasurement of transmembrane potentials. In particular, the presentinvention is directed to compositions and optical methods fordetermining transmembrane potentials across the plasma membrane ofbiological cells.

[0003] Fluorescence detection and imaging of cellular electricalactivity is a technique of great importance and potential (Grinvald, A.,Frostig, R. D., Lieke, E., and Hildesheim, R. 1988. Optical imaging ofneuronal activity. Physiol. Rev. 68:1285-1366; Salzberg, B. M. 1983.Optical recording of electrical activity in neurons using molecularprobes. In Current Methods in Cellular Neurobiology. J. L. Barker,editor. Wiley, N.Y. 139-187; Cohen, L. B. and S. Lesher. 1985. Opticalmonitoring of membrane potential: methods of multisite opticalmeasurement. In Optical Methods in Cell Physiology. P. de Weer and B. M.Salzberg, editors. Wiley, N.Y. 71-99).

[0004] Mechanisms for optical sensing of membrane potential havetraditionally been divided into two classes:

[0005] (1) sensitive but slow redistribution of permeant ions from theextracellular medium into the cell, and

[0006] (2) fast but small perturbations of relatively impermeable dyesattached to one face of the plasma membrane. see, Loew, L. M., “How tochoose a potentiometric membrane probe”, In Spectroscopic MembraneProbes. L. M. Loew, ed., 139-151 (1988) (CRC Press, Boca Raton); Loew,L. M., “Potentiometric membrane dyes”, In Fluorescent and LuminescentProbes for Biological Activity. W. T. Mason, ed., 150-160 (1993)(Academic Press, San Diego).

[0007] The permeant ions are sensitive because the ratio of theirconcentrations between the inside and outside of the cell can change byup to the Nernstian limit of 10-fold for a 60 mV change in transmembranepotential. However, their responses are slow because to establish newequilibria, ions must diffuse through unstirred layers in each aqueousphase and the low-dielectric-constant interior of the plasma membrane.Moreover, such dyes distribute into all available hydrophobic bindingsites indiscriminately. Therefore, selectivity between cell types isdifficult. Also, any additions of hydrophobic proteins or reagents tothe external solution, or changes in exposure to hydrophobic surfaces,are prone to cause artifacts. These indicators also fail to give anyshift in fluorescence wavelengths or ratiometric output. Suchdual-wavelength readouts are useful in avoiding artifacts due tovariations in dye concentration, path length, cell number, sourcebrightness, and detection efficiency.

[0008] By contrast, the impermeable dyes can respond very quicklybecause they need little or no translocation. However, they areinsensitive because they sense the electric field with only a part of aunit charge moving less than the length of the molecule, which in turnis only a small fraction of the distance across the membrane.Furthermore, a significant fraction of the total dye signal comes frommolecules that sit on irrelevant membranes or cells and that dilute thesignal from the few correctly placed molecules.

[0009] In view of the above drawbacks, methods and compositions areneeded which are sensitive to small variations in transmembranepotentials and can respond both to rapid, preferably on a millisecondtimescale, and sustained membrane potential changes. Also needed aremethods and compositions less susceptible to the effects of changes inexternal solution composition, more capable of selectively monitoringmembranes of specific cell types, and providing a ratiometricfluorescence signal. This invention fulfils this and related needs.

SUMMARY OF THE INVENTION

[0010] Methods and compositions are provided for determiningtransmembrane electrical potential (membrane potential), particularlyacross the outermost (plasma) membrane of living cells. In one aspect,the method comprises:

[0011] (a) introducing a first reagent comprising a hydrophobicfluorescent ion, which is capable of redistributing from a first face ofthe membrane to a second face of the membrane in response to changes inthe potential of the membrane, as described by the Nernst equation,

[0012] (b) introducing a second reagent which labels one face, usuallythe extracellular face of the membrane, which second reagent comprises achromophore, capable of undergoing energy transfer by either (i)donating excited state energy to the fluorescent ion, or (ii) acceptingexcited state energy from the fluorescent ion,

[0013] (c) exposing the membrane to excitation light of an appropriatewavelength, typically in the ultraviolet or visible region;

[0014] (d) measuring energy transfer between the fluorescent ion and thesecond reagent, and

[0015] (e) relating the extent of energy transfer to the membranepotential.

[0016] The second reagent is preferably a fluorophore. In each case theexcited state interaction can proceed by fluorescence resonance energytransfer (FRET), which is preferred, or some other mechanism such aselectron transfer, exchange (Dexter) interaction, paramagneticquenching, or promoted intersystem crossing. The method finds particularutility in detecting changes in membrane potential of the plasmamembrane in biological cells.

[0017] Preferably, the hydrophobic ion is an anion which labels theextracellular face of the plasma membrane. Upon addition of thehydrophobic fluorescent anion to the membrane, cell, or tissuepreparation, the anion partitions into the plasma membrane, where itdistributes between the extracellular and intracellular surfacesaccording to a Nernstian equilibrium. Changes in the membrane potentialcause the fluorescent anion to migrate across the membrane so that itcan continue to bind to whichever face (the intracellular orextracellular face) is now positively charged. Since the efficiency ofenergy transfer between the two reagents is a function of the distancebetween them and this distance varies as the fluorescent anionredistributes back and forth across the membrane, measurement of energytransfer provides a sensitive measure of changes in the transmembranepotential. For example, if the membrane potential (intracellularrelative to extracellular) changes from negative to positive, thefluorescent hydrophobic anion is pulled from the extracellular surfaceto the intracellular surface of the plasma membrane. If the secondreagent is one which is on the extracellular face, this results in anincrease in the distance between the anion and the second reagent and aconcomitant decrease in the efficiency of FRET and quenching between thetwo species. Thus, fluorescence measurements at appropriate excitationand emission wavelengths provide a fluorescent readout which issensitive to the changes in the transmembrane potential. Typically, thetime constant for the redistribution of the fluorescent anion is rapidand in the millisecond time scale thus allowing the convenientmeasurement both of rapid cellular electrical phenomena such as actionpotentials or ligand-evoked channel opening, as well as slower and moresustained changes evoked by altering the activity of ion pumps orexchangers.

[0018] Conventional electrophysiological techniques read the potentialat the tip of an electrode and are thus limited to measurements of asingle cell. By contrast, the optical indicators described herein areparticularly advantageous for monitoring the membrane potential of manyneurons or muscle cells simultaneously. Optical indicators, unlikeconventional microelectrodes, do not require physical puncture of themembrane; in many cells or organelles, such puncture is highly injuriousor mechanically difficult to accomplish. Optical indicators are thussuitable for cells too small or fragile to be impaled by electrodes.

[0019] In another aspect of the invention, the voltage sensing methodsallow one to detect the effect of test samples, such as potentialtherapeutic drug molecules, on the activation/deactivation of iontransporters (channels, pumps, or exchangers) embedded in the membrane.

[0020] The compositions of the present invention comprise two reagents.The first reagent comprises a hydrophobic fluorescent ion (preferably ananion) which is capable of redistributing from one face of a membrane tothe other in response to changes in transmembrane potential. This anionis referred to as the mobile or hydrophobic anion. Exemplary anions arepolymethine oxonols, tetraaryl borates conjugated to fluorophores andfluorescent complexes of rare earth and transition metals. The secondreagent comprises a chromophore, preferably a fluorophore, capable ofundergoing energy transfer by either (i) donating excited state energyto the fluorescent anion, or (ii) accepting excited state energy fromthe fluorescent anion. The second reagent binds selectively to one faceof the membrane, and unlike the first reagent, does not redistribute inresponse to transmembrane potential changes. Therefore, it is referredto as the asymmetrically bound or immobile reagent. Exemplary secondreagents include fluorescent lectins, fluorescent lipids, fluorescentcarbohydrates, fluorescently labelled antibodies against surfacemembrane constituents, and amphiphilic fluorescent dyes. In certainpreferred embodiments of the invention, the first and second reagentsare bound together by a suitable flexible linker group. The linker groupis long enough to permit the first reagent to reside in the oppositeface of the membrane from the second reagent and reduce FRET.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The invention may be better understood with reference to theaccompanying drawings, in which:

[0022]FIGS. 1A and 1B illustrate a scheme of the voltage-sensitive FRETmechanism;

[0023]FIG. 2 illustrates normalized excitation and emission spectra for(A) fluorescein-labeled wheat germ agglutinin (FL-WGA) in Hanks'Balanced Salt Solution (HBSS), (B)(1,3-dihexyl-2-thiobarbiturate)trimethine oxonol [DiSBA-C₆-(3)] inoctanol, and (C) TR-WGA in HBSS;

[0024]FIG. 3 illustrates displacement currents of 2.3 μM DiSBA-C₆-(3) inL-M(TK⁻) cells at 20° C.;

[0025]FIG. 4 illustrates voltage dependence of DiSBA-C₆-(3) moved duringthe displacement and tailcurrents for step voltage changes from a −30 mVholding potential;

[0026]FIG. 5 illustrates voltage dependence of DiSBA-C₆-(3) displacementcurrent time constants in L-M(TK⁻) cells for the same data shown in FIG.4;

[0027]FIG. 6 illustrates simultaneous fluorescence changes of theFL-WGA/DiSBA-C₄-(3) pair in response to 4 depolarizations from −70 mV of40, 80, 120, and 160 mV in a L-M(TK⁻) cell at 20° C., with the singlewavelength fluorescence emission traces of DiSBA-C₄-(3) and FL-WGA beingshown in panels A and B, respectively, and the FL-WGA/DiSBA-C₄-(3) ratiodisplayed in (C);

[0028]FIG. 7 illustrates the time course of the fluorescence change ofthe FL-WGA/DiSBA-C₁₀-(3)pair in response to a 100 mV depolarization from−70 mV;

[0029]FIG. 8 illustrates a single sweep trace of fluorescence ratiochanges from the FL-WGA/DiSBA-C₄-(3) pair in beating neonatal cardiacmyocytes, with the top trace (A) showing the FL-WGA channel, (B) thelonger wavelength oxonol channel and (C) the FL-WGA/oxonol ratio, inwhich motion artifacts are significantly reduced; and

[0030]FIG. 9 illustrates the fluorescence changes of theFL-WGA/DiSBA-C₆-(3) pair in a voltage clamped astrocytoma cell, the toptrace (A) being the DiSBA-C₆-(3) emission, (B) the FL-WGA fluorescencesignal and (C) the FL-WGA/oxonol ratio.

[0031]FIG. 10 shows the synthesis of a fluorescent tetraaryl borate.

[0032]FIG. 11 shows a synthesis of an asymmetric oxonol and its linkageto a second reagent.

[0033]FIG. 12 shows possible linkage points (X) of oxonols to a secondreagent.

[0034]FIG. 13 shows a synthesis of Di-SBA-C₆-(3).

[0035]FIG. 14 shows the synthesis of a bifunctional linker.

[0036]FIGS. 15 and 16 show the synthesis of an asymmetric oxonol with alinker suitable for attachment to a second reagent.

[0037]FIG. 17 shows FRET between Cou-PE, a conjugate of a6-chloro-7-hydroxycoumarin to dimyristoylphosphatidylethanolamine, asFRET donor, to a bis-(1,3-dihexyl-2-thiobarbiturate)-trimethineoxonol inan astrocytoma cell.

[0038]FIG. 18 shows FRET between DMPE-glycine-coumarin (Cou-PE) as FRETdonor to a bis-(1,3-dihexyl-2-thiobarbiturate)-trimethineoxonol inL-cells.

[0039]FIG. 19 shows FRET between DMPE-glycine-coumarin (Cou-PE) as FRETdonor to a bis-(1,3-dihexyl-2-thiobarbiturate)-trimethineoxonol incardiomyocytes measured by ratio ouput.

[0040]FIG. 20 shows representative fluorescent phosphatidylethanolamineconjugates that function as FRET donors to the oxonols. The structureson the left depict representative fluorophores and X denotes the site ofattachment of the phosphatidylethanolamine (PE). The structure (PE-R) onthe right shows a phosphatidylethanolamine where R denotes a fluorophoreattached to the amine of the ethanolamine.

[0041]FIG. 21 shows (A) emission spectrum of the Cou-PE; (B) theexcitation spectrum of DiSBA-C₆-(5); and (C) the emission spectrum ofDiSBA-C₆-(5).

[0042]FIG. 22 shows the speed of DiSBA-C₆(5) translocation in responseto a 100 mV depolarization step, using FRET from asymmetrically labeledCou-PE.

[0043]FIG. 23 shows the spectra of [Tb (Salen)₂]⁻¹ (top) and [Eu(Salen)₂]⁻¹ (bottom), both as piperidinium salts dissolved inacetonitrile.

DESCRIPTION OF THE PREFERRED EMBODIMENT

[0044] The following definitions are set forth to illustrate and definethe meaning and scope of the various terms used to describe theinvention herein.

[0045] The term “hydrocarbyl” shall refer to an organic radicalcomprised of carbon chains to which hydrogen and other elements areattached. The term includes alkyl, alkenyl, alkynyl and aryl groups,groups which have a mixture of saturated and unsaturated bonds,carbocyclic rings and includes combinations of such groups. It may referto straight chain, branched-chain, cyclic structures or combinationsthereof.

[0046] The term “alkyl” refers to a branched or straight chain acyclic,monovalent saturated hydrocarbon radical of one to twenty carbon atoms.

[0047] The term “alkenyl” refers to an unsaturated hydrocarbon radicalwhich contains at least one carbon-carbon double bond and includesstraight chain, branched chain and cyclic radicals.

[0048] The term “alkynyl” refers to an unsaturated hydrocarbon radicalwhich contains at least one carbon-carbon triple bond and includesstraight chain, branched chain and cyclic radicals.

[0049] The term “lower” referred to herein in connection with organicradicals or compounds respectively defines such with up to and includingsix, preferably up to and including four carbon atoms. Such groups maybe straight chain or branched.

[0050] The term “heteroalkyl” refers to a branched or straight chainacyclic, monovalent saturated radical of two to forty atoms in the chainin which at least one of the atoms in the chain is a heteroatom, suchas, for example, oxygen or sulfur.

[0051] The term “lower-alkyl” refers to an alkyl radical of one to sixcarbon atoms. This term is further exemplified by such radicals asmethyl, ethyl, n-propyl, isopropyl, isobutyl, sec-butyl, n-butyl andtert-butyl, n-hexyl and 3-methylpentyl.

[0052] The term “cycloalkyl” refers to a monovalent saturatedcarbocyclic radical of three to twelve carbon atoms in the carbocycle.

[0053] The term “heterocycloalkyl” refers to a monovalent saturatedcyclic radical of one to twelve atoms in the ring, having at least oneheteroatom, such as oxygen or sulfur) within the ring.

[0054] The term “alkylene” refers to a fully saturated, cyclic oracyclic, divalent, branched or straight chain hydrocarbon radical of oneto forty carbon atoms. This term is further exemplified by radicals suchas methylene, ethylene, n-propylene, 1-ethylethylene, and n-heptylene.

[0055] The term “heteroalkylene” refers to an alkylene radical in whichat least one of the atoms in the chain is a heteroatom.

[0056] The term “heterocyclo-diyl” refers to a divalent radicalcontaining a heterocyclic ring. The free valences may both be on theheterocyclic ring or one or both may be on alkylene substituentsappended onto the ring.

[0057] The term “lower-alkylene” refers to a fully saturated, acyclic,divalent, branched or straight chain hydrocarbon radical of one to sixcarbon atoms. This term is further exemplified by such radicals asmethylene, ethylene, n-propylene, i-propylene, n-butylene, i-butylene(or 2-methylpropylene), isoamylene (or 3,3 dimethylpropylene),pentylene, and n-hexylene.

[0058] The term “cycloalkyl lower-alkyl” refers to a cycloalkyl groupappended to a lower-alkyl radical. This term is exemplified by, but notlimited to, groups such as cyclopropylmethyl, cyclopentylmethyl,cyclopentylethyl, and cyclopentylpropyl.

[0059] The term “substituted phenyl” refers to a phenyl group which ismono-, di-, tri-,or tetra-substituted, independently, with hydrocarbyl,alkyl, lower-alkyl, cycloalkyl or cycloalkyl-lower alkyl.

[0060] The term “aryl” refers to an aromatic monovalent carbocyclicradical having a single ring (e.g., phenyl) or multiple condensed rings(e.g., naphthyl, anthracenyl), which can optionally be mono-, di-, ortri-substituted, independently, with hydrocarbyl, alkyl, lower-alkyl,cycloalkyl or cycloalkyl lower alkyl.

[0061] The term “arylene” refers to an aromatic divalent carbocyclicradical. The open valence positions may be at any position on thering(s). In the case of a divalent phenyl radical, they may be ortho,meta or para to each other.

[0062] The term “aralkyl” refers to an aryl group appended to alower-alkyl radical. This term is exemplified by, but not limited to,groups such as benzyl, 2-phenylethyl and 2-(2-naphthylethyl).

[0063] The term “aralkenyl” refers to an aryl group appended to a fullyconjugated alkenyl radical. This term is exemplified by styrenyl (cisand trans) and 1-phenyl butadienyl and 1-naphthyl butadienyl (includingall possible combinations of the Z and E isomers about the doublebonds).

[0064] The term “halo” refers to fluoro, bromo, chloro and iodo.

[0065] The term “lower-alkylthiol” refers to the group R—S—, where R islower-alkyl.

[0066] The term “leaving group” means a group capable of being displacedby a nucleophile in a chemical reaction, for example halo, alkylsulfonates (e.g., methanesulfonate), aryl sulfonates, phosphates,sulfonic acid, sulfonic acid salts, imidazolides, N-hydroxy succinimidesand the like.

[0067] The term “linker” refers to any chemically and biologicallycompatible covalent grouping of atoms which can serve to link togetherthe first and second reagents of this invention. Generally, preferredlinkers have from 20 to 40 bonds from end to end, preferably 25 to 30bonds, and may be branched or straight chain or contain rings. The bondsmay be carbon-carbon or carbon-heteroatom or heteroatom-heteroatombonds. The linkage can be designed to be hydrophobic or hydrophilic. Thelinking group can contain single and/or double bonds, 0-10 heteroatoms(O, S preferred), and saturated or aromatic rings. The linking group maycontain groupings such as ester, ether, sulfide, disulfide and the like.

[0068] The term “amphiphilic” refers to a molecule having both ahydrophilic and a hydrophobic portion.

[0069] Methods and compositions are provided for detecting changes inmembrane potential biological systems. One aspect of the detectionmethod comprises:

[0070] (a) introducing a first reagent comprising a hydrophobicfluorescent anion capable of redistributing from a first face of themembrane to a second face of the membrane in response to changes in thepotential of the membrane,

[0071] (b) introducing a second reagent which labels the first face orthe second face of the membrane, which second reagent comprises afluorophore capable of undergoing energy transfer by either (i) donatingexcited state energy to the fluorescent anion, or (ii) accepting excitedstate energy from the fluorescent anion,

[0072] (c) exposing the membrane to excitation light of appropriatewavelengths;

[0073] (d) measuring energy transfer between the fluorescent anion andthe second reagent, and

[0074] (e) relating the energy transfer to the change in plasma membranepotential.

[0075] The preferred mode of energy transfer is fluorescence resonanceenergy transfer (FRET). The method finds particular utility in detectingchanges in membrane potential of the plasma membrane in biologicalcells.

[0076] The compositions used in the methods of the invention comprisetwo reagents. The first reagent comprises a hydrophobic fluorescentanion which rapidly redistributes from one face of the plasma membraneto the other in response to changes in transmembrane potential. Thisspecies is referred to as the mobile or hydrophobic anion. The secondreagent comprises a fluorophore capable of undergoing energy transfer byeither (i) donating excited state energy to the fluorescent anion, or(ii) accepting excited state energy from the fluorescent anion,typically by FRET.

[0077] The first and second reagents are spectroscopically complementaryto each other, by which is meant that their spectral characteristics aresuch that excited state energy transfer can occur between them. Eitherreagent can function as the donor or the acceptor, in which case theother reagent is the corresponding complement, i.e., the acceptor ordonor respectively. Both FRET and quenching are highly sensitive to thedistance between the two species. For example, the nonradiativeForster-type quenching observed in FRET varies inversely with the sixthpower of the distance between the donor and acceptor species. Therefore,when the membrane potential changes and the hydrophobic fluorescentanion moves either further away from or closer to the second reagent,FRET between the two reagents is either reduced or enhancedsignificantly. Other mechanisms such as electron-transfer, Dexterexchange interaction, paramagnetic quenching, and promoted intersystemcrossing are even shorter-range and require the two reagents to collideor at least come within 1 nm of each other.

[0078] Previously reported voltage-sensitive fluorescent indicatorsoperating by potential-driven redistribution of the fluorophore acrossthe membrane had response times of >100 ms, often as long as minutes.One aspect of the present invention provides highly fluorescent anionicdyes which translocate across the membrane at much faster rates, withexponential time constants typically less than about 10 ms, frequentlyless than 5 ms, most frequently from about 1 to 3 ms and most preferablyless than 1 ms time scale (e.g., 0.1 to 1 ms). These translocation ratesare independent of the presence of the second reagent on theextracellular surface of the membrane. Response times of <1 ms arenecessary for accurate measurement of single action potentials inindividual neurons and are obtained with some of the dyes describedherein (e.g., hexyl-substituted pentamethineoxonol, diSBA-C₆-(5)). Otherdyes described herein have response times in the 2-5 ms range, which arefast enough to monitor voltage changes in heart and smooth muscle, manysynaptic potentials in single neurons, and the average firing activityin populations of neurons (for example, mapping the electrical responsesof different regions of the central nervous system to sensory inputs).The indicators of the present invention are also able to follow slowervoltage changes over a time scale of seconds to minutes.

I. FIRST REAGENT—HYDROPHOBIC FLUORESCENT ANIONS

[0079] In the compositions and methods of the present invention, thefirst reagent comprises a hydrophobic ion (fluorescence donor, acceptor,or quencher) which serves as a voltage sensor and moves within themembrane from one face of the membrane to another in response to changesin the transmembrane potential. The distribution of hydrophobic ionsbetween the two membrane-aqueous interfaces (the extracellular interfaceand the intracellular interface) is determined by the membranepotential. Cations will tend to congregate at the negatively chargedmembrane interface and correspondingly, anions will move to thepositively charged interface. The inherent sensitivity of the inventionis based on the large interfacial concentration changes of the mobileion at physiologically relevant changes in membrane potentials.Potentially, a 60 mV change produces 10-fold change in the ratio of theanion concentrations at the respective interfaces. The methods of thisinvention couple this change in interfacial concentration to anefficient fluorescent readout thus providing a sensitive method ofdetecting changes in transmembrane potential. The speed of thefluorescence change is dependent on the membrane translocation rate ofthe hydrophobic ion.

[0080] Preferably, the fluorescent ions which translocate across theplasma membrane are hydrophobic in order to bind strongly to the plasmamembrane and translocate rapidly across it in response to changes intransmembrane potential. Preferably, the ion will have a single chargewhich will be delocalized across a significant portion of the dye,preferably the entire dye. Delocalization of the charge reduces the Borncharging energy (inversely proportional to anion radius) required tomove a charged molecule from a hydrophilic to a hydrophobic environmentand facilitates rapid translocation of ions (Benz, R. 1988. “Structuralrequirement for the rapid movement of charged molecules acrossmembranes”, Biophy. J. 54:25-33). Increasing hydrophobicity minimizesrelease of the bound dye from the plasma membrane and buries the iondeeper into the membrane, which decreases the electrostatic activationenergy for translocation. Polar groups on the ion should be kept to aminimum and shielded as much as possible to disfavor salvation in theheadgroup region of the bilayer. However, hydrophobicity cannot beincreased without limit, because some aqueous solubility is required topermit cellular loading. If necessary, dyes may be loaded with the aidof amphiphilic solubilizing reagents such as beta-cyclodextrin,Pluronics such as Pluronic F-127, or polyethylene glycols such asPEG400, which help solubilize the hydrophobic ions in aqueous solution.

[0081] The term “hydrophobic” when used in the context of thehydrophobic ion refers to a species whose partition coefficient betweena physiological saline solution (e.g. HBSS) and octanol is preferably atleast about 50, and more preferably at least about 1000. Its adsorptioncoefficient to a phospholipid bilayer (such as for example a membranederived from a human red blood cell is at least about 100 nm, preferablyat least about 300 nm (where the membrane is 3 nm). Methods ofdetermining partition coefficients and adsorption coefficients are knownto those of skill in the art.

[0082] It is generally preferred that the hydrophobic dye be an anionicspecies. Ester groups of biological membranes generate a sizable dipolepotential within the hydrocarbon core of the membrane. This potentialaids anion translocation through the hydrophobic layer but hinderscations. Therefore, where membrane translocation is concerned, anionshave a tremendous inherent speed advantage over cations. For example, itis known that for the isostructural ions tetraphenylphosphonium cationand tetraphenylborate anion, the anion is much more permeable than thecation (Flewelling, R. F. and Hubbell, W. L. 1986. “The membrane dipolepotential in a total membrane potential model”, Biophys. J. 49:541-552).

[0083] Preferably, the anions should be strongly fluorescent whenadsorbed to the membrane, whereas they should have minimal fluorescencewhen free in aqueous solution. Preferably, the anionic fluorophoresshould be at least four times, and more preferably at least about eighttimes, brighter when adsorbed to the membrane. In the case of thethiobarbiturate oxonols described herein, their fluorescence is about 20fold greater in the membrane than in water. In principle, if the dyebound extremely tightly to the membrane one would not need a high ratioof fluorescence when bound to the membrane to that when free in aqueoussolution; however, because in reality the volume of the membrane is tinyrelative to the aqueous solution and some water solubility is necessaryfor loading of the dye into cells and tissue, it is desirable for thefirst reagent to be at least about four times more strongly fluorescentin a membrane than in aqueous solution.

[0084] The anions also should not act as ionophores, especiallyprotonophores, since such behavior may generate sustained leakagecurrents. Therefore, the protonation pKa of the anion is typically wellbelow 7, preferably below 5, more preferably below 3. Red to infra-redwavelengths of excitation and emission are preferred to avoid tissuescattering and heme absorbances. Photodynamic damage should be kept aslow as possible, probably best by minimizing triplet state formation andthe resulting generation of singlet oxygen.

[0085] The fluorescent hydrophobic ions include polymethine oxonols,tetraaryl borates conjugated to fluorophores and fluorescent complexesof rare earth and transition metals.

[0086] A. Polymethine Oxonols

[0087] The term “polymethine oxonol” refers to molecules comprising twopotentially acidic groups linked via a polymethine chain and possessinga single negative charge delocalized between the two acidic groups. Thepreferred acidic groups are barbiturates or thiobarbiturates. They maybe symmetric or asymmetric, i.e., each of the two (thio)barbiturates maybe the same or different. The symmetric (thio)barbiturate oxonols aredescribed by the conventional shorthand DiBA-C_(n)-(x) andDiSBA-C_(n)-(x), where DiBA refers to the presence of two barbiturates,DiSBA refers to the presence of two thiobarbiturates, C_(n) representsalkyl substituents having n carbon atoms on the nitrogen atoms of the(thio)barbiturates, and x denotes the number of carbon atoms in thepolymethine chain linking the (thio)barbiturates. It has beenunexpectedly found that oxonols with long chain alkyl substituents (e.g.C_(n) greater than hexyl, especially decyl in the pentamethine oxonols)translocate surprisingly rapidly across plasma membranes.

[0088] An extremely useful property of these oxonols is that theirfluorescence emission maximum at 560 nm is 20 times brighter when boundto membranes than in aqueous solution [Rink, T. J., Montecucco, C.,Hesketh, T. R., and Tsien, R. Y. 1980. Lymphocyte membrane potentialassessed with fluorescent probes. Biochim. Biophys. Acta 595:15-30].Furthermore, the is negative charge is delocalized throughout thechromophore with the four equivalent oxygens containing the majority ofthe charge. The high electron affinity of the thiobarbiturate moietiesdiscourages protonation, pKa<1, and resists photooxidative bleaching.The four N-alkyl groups and the thiocarbonyl give the molecule anecessary amount of hydrophobicity needed for tight membrane binding andrapid translocation.

[0089] Oxonol compounds used in this invention have a general structureof Formula I.

[0090] wherein:

[0091] R is independently selected from the group consisting of H,hydrocarbyl and heteroalkyl;

[0092] X is independently oxygen or sulfur; and

[0093] n is an integer from 1 to 3;

[0094] and salts thereof.

[0095] The oxonol anions are usually loaded as salts with the cationtypically being H⁺, alkali metal, substituted ammonium, or pyridinium.

[0096] Preferably X is sulfur, i.e., the hydrophobic anion is abis-(1,3-dialkyl-2-thiobarbiturate)-polymethine oxonol or a derivativethereof.

[0097] When R is a hydrocarbyl group, it can be independently selectedfrom the group consisting of alkyl, aryl, aralkyl, cycloalkyl andcycloalkyl lower-alkyl. Typically these groups have from about 4 toabout 40 carbon atoms, more preferably, about 5 to about 20 carbonatoms. Aryl groups can be substituted with hydrocarbyl, alkyl, loweralkyl, heteroalkyl and halogen groups. Oxonols in which the R groups ona particular (thio)barbiturate moiety are different to each other arespecifically contemplated by this invention and can be prepared fromunsymmetrical urea derivatives.

[0098] In some embodiments, R is a hydrocarbyl group of the formula:

—(CH₂)_(p)(CH═CH—CH₂)_(q)(CH₂)_(r)CH₃

[0099] wherein:

[0100] p is an integer from 1 to about 20 (preferably about 1 to 2);

[0101] q is an integer from 1 to about 6, preferably 1 to 2;

[0102] the stereochemistry of the double bond(s) may be cis or trans,cis being preferred;

[0103] r is an integer from 1 to about 20 (preferably about 1 to 3), andp+3q+r+1 40, preferably from about 4 to 20, more preferably about 6 to10.

[0104] In another embodiment of the polymethine oxonols, R is aheteroalkyl group of the formula:

—(CH₂)_(x)A_(y)(CH₂)_(z)CH₃,

[0105] wherein:

[0106] A is oxygen or sulfur;

[0107] x is independently an integer from 1 to about 20 (preferablyabout from about 10 to 15);

[0108] y is independently 0 or 1;

[0109] z is independently an integer from 1 to about 20 (preferablyabout 10 to 15); and

[0110] x+y+z 40, preferably x+y+z=an integer from about 5 to 25, morepreferably about 5 to 10.

[0111] In other embodiments, R is a phenyl group independentlysubstituted with up to four substituents selected from the groupconsisting of hydrocarbyl, heteroalkyl, halogen and H.

[0112] In other embodiments, one of the four R groups incorporates alinker to the second reagent, as described below in section III.

[0113] An oxonol's negative charge is distributed over the entire thechromophore. Bis(thiobarbiturate)trimethineoxonols absorb at 542 nm(ext. coefficient=200,000 M-⁻¹ cm⁻¹), emit at 560 nm and have a quantumyield of 0.4 in octanol. An oxonol where R=n-hexyl, DiSBA-C₆-(3),translocates with a time constant (τ)<3 ms in voltage clamped mammaliancells. The corresponding decyl compound, DiSBA-C₁₀-(3), translocateswith a time constant<2 ms. The molecular requirement for rapidtranslocation is nicely met with the symmetric oxonols.Bis(thiobarbiturate)pentamethineoxonols absorb at −630 nm and emit at−660 nm. The negative charge is further delocalized in such red-shiftedoxonols. As expected, the translocation rates for the pentamethineoxonols are faster than for the trimethine oxonols. DiSBA-C₄-(5) crossesthe membrane with τ<3 ms, six times faster than the correspondingtrimethine oxonol. DiSBA-C₆-(5) translocates with τ ⁻0.4 ms at 20.

[0114] B. Tetraaryl Borate—Fluorophore Conjugates

[0115] Another useful class of fluorescent hydrophobic anions aretetraaryl borates having a general structure of Formula II.

[(Ar¹)₃B—Ar²—Y—FLU]⁻  Formula II

[0116] wherein:

[0117] Ar¹ is an aryl group;

[0118] Ar² is a bifunctional arylene group;

[0119] B is boron;

[0120] Y is oxygen or sulfur; and

[0121] FLU is a neutral fluorophore.

[0122] Frequently Ar¹ is substituted with one or two electronwithdrawing groups, such as but not limited to CF₃. In selectedembodiments, Ar¹ and Ar² are optionally substituted phenyl groups asshown below for the structure of Formula III.

[0123] wherein:

[0124] each R′ is independently H, hydrocarbyl, halogen,

[0125] CF₃ or a linker group;

[0126] n is an integer from 0 to 5;

[0127] each X is independently H, halogen or CF₃;

[0128] m is an integer from 0 to 4;

[0129] Y is oxygen or sulfur; and

[0130] FLU is a neutral fluorophore.

[0131] When R′ is hydrocarbyl, it is typically from 1 to about 40 carbonatoms, preferably 3 to about 20 carbon atoms, more preferably about 5 to15 carbon atoms. Preferably, R′ is a lower alkyl group, more preferably(for ease of synthesis) all the R′s are H. When R′ is not hydrocarbyl,it is frequently CF₃ and n=1. In selected embodiments X=F and m=4. X istypically electron-withdrawing to prevent photoinduced electron transferfrom the tetraaryl borate to the fluorophore, which quenches the latter.X=F is most preferred.

[0132] 1. Synthesis of Tetraaryl Borate—Fluorophore Coniugates

[0133] A general synthesis of fluorescent tetraaryl borate anions hasbeen developed and is shown in FIG. 10 for an exemplary fluorescentbimane tetraaryl borate conjugate (identified as Bormane, compound IV inFIG. 10).

[0134] In general terms, a triaryl borane is reacted with a protectedphenoxy or thiophenoxy organometallic reagent, such as, for example, anorganolithium derivative. The protecting group is subsequently removedand the unmasked phenol (or thiophenol) is reacted with a fluorophorebearing a leaving group. Nucleophilic displacement of the leaving groupfollowed by conventional purification of the crude reaction productfurnishes the tetraaryl borate anion conjugated to the fluorophore.Substituents R′ and X are varied by appropriate choice of the startingtriaryl borane and the phenoxy (or thiophenoxy) organometallic. Suitablestarting materials can be obtained from Aldrich Chemical Co.(Milawaukee, Wis.) and other commercial suppliers known to those ofskill in the art. Thus in these species, a fluorophore is conjugated toa functionalized borate core. This general synthetic method allows oneto attach any fluorophore to the borate anion.

[0135] 2. Neutral Fluorophores

[0136] As polar chromophores retard the membrane translocation rate, itis preferred that the fluorophore conjugated to the tetraaryl borate bea neutral species. For purposes of the present invention, a neutralfluorophore may be defined as a fluorescent molecule which does notcontain charged functional groups. Representative fluorescent moleculesbearing leaving groups and suitable for conjugation are available fromMolecular Probes (Portland, Oreg.), Eastman Kodak (Huntington, Tenn.),Pierce Chemical Co. (Rockville, Md.) and other commercial suppliersknown to those of skill in the art. Alternatively, leaving groups can beintroduced into fluorescent molecules using methods known to those ofskill in the art.

[0137] Particularly suitable classes of neutral fluorophores which canbe conjugated to the tetraaryl borates for use in accordance with thepresent invention include, but are not limited to, the following:bimanes; bodipys; and coumarins.

[0138] Bodipys (i.e., difluoroboradiazaindacenes) may be represented bya general structure of Formula IV.

[0139] wherein:

[0140] each R¹, which may be the same or different, is independentlyselected from the group consisting of H, lower alkyl, aryl,heteroaromatic, aralkenyl and an alkylene attachment point;

[0141] each R², which may be the same or different, is independentlyselected from the group consisting of H, lower alkyl, phenyl and analkylene attachment point.

[0142] For the purposes of this disclosure, the term “alkyleneattachment point” refers to the group —(CH₂)_(t)— or —(CH₂)_(t)—C(O)—wherein, t is an integer from 1 to 10, and one valence bond is attachedto the fluorophore and the other valence bond is attached to thetetraaryl borate. Preferably, t=1, i.e. the alkylene attachment point isa methylene group. As will be apparent to one of skill in the art, allfluorophores will possess one attachment point at which they will beconjugated to the tetraaryl borate. Generally, the precursor moleculeused to conjugate the fluorophore to the tetraaryl borate will carry aleaving group at the attachment point. Reaction of this precursor withan appropriate nucleophile on the tetraaryl borate (e.g., an amine,hydroxy or thiol), will provide a fluorophore-tetraaryl borate conjugatelinked together at the attachment point. The term “attachment point”refers more broadly to a chemical grouping which is appropriate to reactwith either a fluorophore or a bifunctional linker to form thefluorescent conjugates and/or linked first and second reagents asdisclosed herein. Frequently, these attachment points will carry leavinggroups, e.g., alkyl tosylates, activated esters (anhydrides,N-hydroxysuccinimidyl esters and the like) which can react with anucleophile on the species to be conjugated. One of skill will recognizethat the relative positioning of leaving group and the nucleophile onthe molecules being linked to each other can be reversed.

[0143] Coumarins and related fluorophores may be represented bystructures of general Formulas V and VI

[0144] wherein:

[0145] each R³, which may be the same or different, is independentlyselected from the group consisting of H, halogen, lower alkyl, CN, CF₃,COOR⁵, CON(R⁵)₂, OR⁵, and an attachment point;

[0146] R₄ is selected from the group consisting of OR⁵ and N(R⁵)₂;

[0147] Z is O, S or NR⁵; and

[0148] each R⁵, which may be the same or different, is independentlyselected from the group consisting of H, lower alkyl and an alkyleneattachment point.

[0149] Bimanes may be represented by a structure of general Formula VII.

[0150] wherein:

[0151] each R⁵, which may be the same or different, is independently H,lower alkyl or an alkylene attachment point.

[0152] Fluorescent tetraaryl borates with coumarins and bimanes attachedhave been prepared. These fluorescent borates translocate with τ<3 ms involtage clamped fibroblasts. Synthesis of an exemplary fluorescenttetraryl borate is described in Example V.

[0153] C. Fluorophore Complexes with Transition Metals

[0154] Lanthanide ions, such as, for example, Tb³⁺ and Eu³⁺, luminescein the green and red regions of the visible spectrum with millisecondlifetimes. The emission is composed of several sharp bands that areindicative of the atomic origin of the excited states. Direct excitationof the ions can be accomplished using deep UV light. Excitation of thelanthanide ions at longer wavelengths is possible when the ions arechelated by absorbing ligands that can transfer excitation energy to theions, which then can luminesce with their characteristic emission as ifthey had been excited directly. Lanthanide complexes of Tb³⁺ and Eu³⁺with absorbing ligands that contribute 4 negative charges, resulting anet charge of −1, may function as mobile ions for the voltage-sensitiveFRET mechanism. The lifetimes of Tb³⁺ and Eu³⁺ are still sufficientlyfast to measure millisecond voltage changes.

[0155] This invention also provides such complexes which can functionthe fluorescent hydrophobic anion (as FRET donors) in the first reagent.Using the ligand bis-(salicylaldehyde)ethylenediamine (Salen)²⁻,[Tb(Salen)₂]⁻¹ and [Eu(Salen)₂]⁻¹ have been made. These complexes absorbmaximally at 350 nm with significant absorbance up to 380 nm andluminesce with the characteristic atomic emission, FIG. 10. The use oflanthanide complexes as donors offers several unique advantages.Scattering, cellular autofluorescence, and emission from directlyexcited acceptors have nanosecond or shorter lifetimes and may berejected by time gating of the emission acquisition (See for exampleMarriott, G., Heidecker, M., Diamandis, E. P., Yan-Marriott, Y. 1994.Time-resolved delayed luminescence image microscopy using an europiumion chelate complex. Biophys. J. 67: 957-965). The elimination of thefast emission reduces the background and gives excellent signal to noiseratios. Another major advantage of using lanthanide chelates as donorsis that the range of FRET is amplified by lateral diffusion in themembrane during the excited state lifetime (Thomas, D. D., Carlsen, W.F., Stryer, L. 1978. Fluorescence energy transfer in the rapid diffusionlimit. Proc. Natl. Acad. Sci. USA 75: 5746-5750). This feature greatlyreduces the need for high concentrations of acceptors to ensureefficient FRET. In addition to reducing the perturbation and stress tothe cellular system from high dye concentrations, the diffusion enhancedFRET will lead to greater voltage sensitivity than is possible in astatic case. Lanthanide chelates can also be used as asymmetricallylabeled donors to mobile acceptors such as the tri and pentamethineoxonols, with the same advantages as discussed above.

[0156] Representative lanthanide complexes which may be used as ahydrophobic fluorescent anion are shown in Formulas XI and XII

[0157] wherein:

[0158] Ln=Tb, Eu, or Sm;

[0159] R is independently H, C1-C8 alkyl, C1-C8 cycloalkyl or C1-C4perfluoroalkyl;

[0160] X and Y are independently H, F, C1, Br, I, NO₂, CF₃, lower(C1-C4) alkyl, CN, Ph, O-(lower alkyl), or OPh; or X and Y together are—CH═CH—; and

[0161] Z=1,2-ethanediyl, 1,3-propanediyl, 2,3-butanediyl,1,2-cyclohexanediyl, 1,2-cyclopentanediyl, 1,2-cycloheptanediyl,1,2-phenylenediyl, 3-oxa-1,5-pentanediyl, 3-aza-3-(loweralkyl)-1,5-pentanediyl, pyridine-2,6-bis(methylene) ortetrahydrofuran-2,5-bis(methylene).

[0162] wherein:

[0163] Ln=Tb, Eu, or Sm;

[0164] R is independently H, C1-C8 alkyl, C1-C8 cycloalkyl or C1-C4perfluoroalkyl;

[0165] X′ and Y′ are independently H, F, C1, Br, I, NO₂, CF₃, lower(C1-C4) alkyl, CN, Ph, O-(lower alkyl), or OPh; or X′ and Y′ togetherare —CH═CH—; and

[0166] Z′ is independently a valence bond, CR₂, pyridine-2-6-diyl ortetrahydofuran-2,5-diyl.

II. IMMOBILE. ASYMMETRICALLY BOUND SECOND REAGENTS

[0167] The second reagent is a fluorescent donor, acceptor, or quencher,complementary to the first reagent, the hydrophobic anion, and bindseither to the extracellular or intracellular face of the plasmamembrane. Thus, the presence of the second reagent on one or the otherface of the membrane desymmetrizes the membrane. As described earlier,irradiation with light of an appropriate wavelength generates afluorescent readout which changes in response to movement of thehydrophobic ion back and forth across the plasma membrane. As would beimmediately apparent to those skilled in the field, there are numerousmolecular species which could function as the fluorescently activedesymmetrizing agent. The primary characteristics for this component arethat it locate on one face of the plasma membrane and function in acomplementary manner (i.e., as a fluorescent donor, acceptor, orquencher) to the hydrophobic ion which shuttles back forth across themembrane as the transmembrane potential changes.

[0168] Exemplary second reagents include fluorescent lectins,fluorescent lipids, fluorescent carbohydrates with hydrophobicsubstituents, fluorescent peptides, fluorescently labelled antibodiesagainst surface membrane constituents, or xanthenes, cyanines andcoumarins with hydrophobic and hydrophilic substituents to promotebinding to membranes and to prevent permeation through membranes.

[0169] A. Fluorescent Lectins

[0170] One class of second reagents are lectins carrying a fluorescentlabel. For purposes of the present invention, a lectin may be defined asa sugar binding protein which binds to glycoproteins and glycolipids onthe extracellular face of the plasma membrane. See, Roth, J., “TheLectins: Molecular Probes in Cell Biology and Membrane Research,” Exp.Patholo. (Supp. 3), (Gustav Fischer Verlag, Jena, 1978). Lectins includeConcavalin A; various agglutinins (pea agglutinin, peanut agglutinin,wheat germ agglutinin, and the like); Ricin, A chain and the like. Avariety of lectins are available from Sigma Chemical Co., St. Louis, Mo.

[0171] Suitable fluorescent labels for use in fluorescent lectinsinclude, but are not limited to, the following: xanthenes (includingfluoresceins, rhodamines and rhodols); bodipys, cyanines, andluminescent transition metal complexes. It will be recognized that thefluorescent labels described below can be used not merely with lectinsbut with the other second reagents described herein. To date, the bestresults with lectins have been obtained with fluorescein labeled wheatgerm agglutinin (FL-WGA).

[0172] 1. Xanthenes

[0173] One preferred class of fluorescent labels comprise xanthenechromophores having a structure of general Formula VIII or IX.

[0174] wherein:

[0175] R⁶ is independently selected from the group consisting of H,halogen, lower alkyl, SO₃H and an alkylene attachment point;

[0176] R⁷ is selected from the group consisting of H, lower alkyl, analkylene attachment point, and R⁸, wherein R⁸ is selected from the groupconsisting of

[0177] wherein:

[0178] each a and a′ is independently selected from the group consistingof H and an alkylene attachment point;

[0179] G is selected from the group consisting of H, OH, OR⁹, NR⁹R⁹ andan alkylene attachment point;

[0180] T is selected from the group consisting of O, S, C(CH₃)₂ and NR⁹;and

[0181] M is selected from the group consisting of O and NR⁹R⁹;

[0182] wherein each R⁹, which may be the same or different, isindependently H or hydrocarbyl.

[0183] 2. Cyanines

[0184] Another preferred class of fluorescent labels are cyanine dyeshaving a structure of general Formula X.

[0185] wherein:

[0186] R¹⁵ is independently selected from the group consisting of H,halogen, lower alkyl, SO₃H, PO₃H₂, OPO₃H₂, COOH, and an alkyleneattachment point;

[0187] R¹⁶ is selected from the group consisting of H, lower alkyl,(CH₂)_(j)COOH, (CH₂)_(j)SO₃H, and an alkylene attachment point; where jis an integer from 1 to 10;

[0188] T′ is selected from the group consisting of O, S, C(CH₃)₂,—CH═CH—, and NR¹⁷, where R¹⁷ is H or hydrocarbyl; and

[0189] n is an integer from 1 to 6.

[0190] B. Fluorescent Lipids

[0191] Fluorescently labeled amphipathic lipids, in particularphospholipids, have also been successfully employed. For purposes of thepresent invention, an amphipathic lipid may be defined as a moleculewith both hydrophobic and hydrophilic groups that bind to but do notreadily cross the cell membrane. Fluorescently labelled phospholipidsare of particular value as second reagent.

[0192] As defined herein, “phospholipids” include phosphatidic acid(PA), and phosphatidyl glycerols (PG), phosphatidylcholines (PC),phosphatidylethanolamines (PE), phospatidylinositols (PI),phosphatidylserines (PS), and phosphatidyl-choline, serine, inositol,ethanolamine lipid derivatives such as egg phosphatidylcholine (EPC),dilauroyl-phosphatidylethanolamine, dimyristoylphosphatidylethanolamine,dipalmitoylphosphatidylethanolamine,distearoylphosphatidyl-ethanolamine, dioleoylphosphatidylethanolamine,distearoyl-phosphatidylserine, dilinoleoyl phosphatidylinositol, andmixtures thereof. They may be unsaturated lipids and may be naturallyoccurring or synthetic. The individual phosphatidic acid components maybe symmtrical, i.e. both acyl residues are the same, or they may beunsymmetrical, i.e., the acyl residues may be different.

[0193] Particularly preferred embodiments include6-chloro-7-hydroxycoumarin-labeled phosphatidylethanolamine (Cou-PE),fluorescein-labeled phosphatidylethanolamine (FL-PE),N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine (NBD-PE),and 5,5′-disulfoindodicarbocyanine-labeled phosphatidylethanolamine(Cy5-PE). The fluorescent group is suitably selected from thosepreviously described as useful in preparing fluorescent lectins.Preferred fluorescent phospholipids include Cou-PE, FL-PE, NBD-PE orCy5-PE. Using Cou-PE as donor with DiSBA-C₆-(3) as acceptor has given a80% ratio change for a 100 mV change in plasma membrane potential, andtypically gives ratio signals twice as large as FL-WGA with the sameoxonol.

[0194] C. Fluorescently Labelled Antibodies

[0195] Antibodies directed against surface antigens such as glycolipidsor membrane proteins can also be fluorescently labeled and used assecond reagents. For example, FITC-labeled antibodies against theglycolipid GD3 stain the outer surface of the melanoma cell line M21 andgive ratio changes up to 10%/100 mV using DiSBA-C₆-(3) as the mobilefluorescent anion. Specificity for particular cell types is likely. tobe easier to achieve with antibodies than with lectins becauseantibodies can be raised against nearly any surface marker. Also,microinjected antibodies could label sites on the cytoplasmic face ofthe plasma membrane, where carbohydrate binding sites for lectins areabsent.

[0196] D. Cytochromes

[0197] Cytochrome c used as a second reagent has also been found tofunction as a quencher which binds to the outer plasma membrane surface.Accordingly, another suitable class of second reagent comprisescytochrome c or apocytochrome c, with or without a fluorescent group aspreviously described in connection with other second reagents.

[0198] E. Fluorescent Carbohydrates

[0199] Yet another preferred class of embodiments of the second reagentincludes fluorescently labeled, amphipathic carbohydrates, e.g.,cyclodextrins which selectively and tightly bind to the extracellularplasma membrane surface. Typically, the carbohydrates are functionalizedwith a hydrophobic tail to facilitate intercalation into the membraneand tight membrane binding. The cyclic sugar imparts good watersolubility for cellular loading and prohibits membrane translocation.Another added benefit is that the cyclodextrins aid the loading of theoxonol.

[0200] F. Fluorescent Pentides and Proteins

[0201] Yet another preferred class of embodiments of the second reagentincludes fluorescently labeled, amphipathic peptides. Typically suchpeptides contain several basic residues such as lysines and arginines tobind electrostatically to negatively charged phospholipid head groups,plus several hydrophobic residues to anchor the peptide to the membrane.Optionally, long-chain alkyl substituents such as N-myristoyl,N-palmitoyl, S-palmitbyl, or C-terminal prenyl groups may providehydrophobicity. The fluorescent label is typically attached via lysineepsilon-amino groups or cysteine sulfhydryl groups.

[0202] Yet another preferred class of embodiments of the second reagentincludes naturally fluorescent proteins such as the Green FluorescentProtein (GFP) of Aequorea victoria (Cubitt, A. B. et al. 1995.Understanding, improving, and using green fluorescent proteins. TrendsBiochem. Sci. 20: 448-455; Chalfie, M., and Prasher, D. C. U.S. Pat No.5,491,084). Such proteins would be fused to native plasma membraneproteins by expression of tandem DNA constructs in which the DNAsequences encoding the GFP and the native protein are concatenated inframe. Alternatively, the GFP could be fused to a motif that causesattachment of glycosylphosphatidylinositol anchors and targeting of theprotein to the plasma membrane.

[0203] As the preceding discussion indicates and would be readilyappreciated by those skilled in the art, a wide variety of knowndonor/acceptor pairs can be used as first and second reagents.Particularly preferred combinations include, but are not limited to, thefollowing, in which the first-named fluorophore is the donor and thesecond is the acceptor: fluorescein/bis-thiobarbituratetrimethineoxonol; Green Fluorescent Protein/bis-thiobarbituratetrimethineoxonol; Green Fluorescent Protein/bis-thiobarbituratepentamethineoxonol; coumarin/bis-thiobarbiturate trimethineoxonol;coumarin/bis-thiobarbiturate pentamethineoxonol; bis-thiobarbituratetrimethineoxonol/Texas Red; bis-thiobarbituratetrimethineoxonol/resorufin; bis-thiobarbiturate trimethineoxonol/Cy5;bis-thiobarbiturate trimethineoxonol/bis-thiobarbituratepentamethineoxonol; Texas Red/bis-thiobarbiturate pentamethineoxonol;NBD/bis-thiobarbiturate trimethineoxonol; NBD/bis-thiobarbituratepentamethineoxonol.

III. LINKER GROUPS BETWEEN FIRST AND SECOND REAGENT

[0204] In some particularly preferred embodiments of the compositionsand methods of the present invention, a linker group is employed betweenthe first and second fluorophores. The linker group maintains a certainminimum proximity between the first and second fluorophores and ensuresefficient energy transfer between the donor and acceptor (or fluorophoreand quencher) when they are on the same side of the membrane, even atlow concentrations. The good energy transfer allows one to separate thedonor emission further from the acceptor absorbance and thus decreasethe spectral crosstalk that contributes to the reduction of thevoltage-sensitive ratio change from theoretical values. Another majoradvantage of a linker is that it converts the system into a unimolecularphenomenon. This greatly simplifies the fluorescence readout, ensures1:1 stoichiometry of donor and acceptor (or fluorophore and quencher),and eliminates the need for optimizing relative loading levels of donorand acceptor for an optimum voltage-sensitive fluorescence change (withthe additional benefit of minimal cellular perturbation and toxicity).The linker group is long enough to span the entire membrane.

[0205] The hydrophobic fluorescent anion and the second reagent areattached to each other by means of a bifunctional linker. “Linker group”shall mean the “chemical arm” between the first and second reagent. Asone skilled in the art will recognize, to accomplish the requisitechemical structure, each of the reactants must contain the necessaryreactive groups.

[0206] Representative combinations of such groups are amino withcarboxyl to form amide linkages, or carboxy with hydroxy to form esterlinkages or amino with alkyl halides to form alkylamino linkages, orthiols with thiols to form disulfides, or thiols with maleimides oralkyl halides to form thioethers. Obviously, hydroxyl, carboxy], aminoand other functionalities, where not present may be introduced by knownmethods. Likewise, as those skilled in the art will recognize, a widevariety of linking groups may be employed. The structure of the linkageshould be a stable covalent linkage formed to attach the two species toeach other. The portions of the linker closest to the second reagent maybe hydrophilic, whereas the portions of the linker closest to the mobilefluorescent anion should be hydrophobic to permit insertion into themembrane and to avoid retarding the voltage-dependent translocation ofthe anion. The covalent linkages should be stable relative to thesolution conditions under which the cells are loaded. Generallypreferred linking groups will comprise 20-40 bonds from one end to theother and 0-10 heteroatoms (NH, O, S) and may be branched or straightchain. Without limiting the foregoing, it should be evident to oneskilled in the art that only combinations of atoms which are chemicallycompatible comprise the linking group. For example, amide, ester,thioether, thioester, keto, hydroxyl, carboxyl, ether groups incombinations with carbon-carbon bonds are acceptable examples ofchemically compatible linking groups. Other chemically compatiblelinkers may be found in U.S. Pat. Nos. 5,470,997 (col. 2 and col. 4-7)and 5,470,843 (cols. 11-13).

[0207] Asymmetric 1,3-substituted thioureas have been prepared for usein synthesizing oxonols with N-substituted linkers containing terminalreactive groups capable of conjugating to appropriate second reagentfluorophores/quenchers. In one example, one of the oxonol substituentsis a pentyl chain (C₅) with a terminal bromide or tosylate group.Thiobarbiturates have been synthesized from these thioureas anddiethylmalonate in ethoxide/ethanol. Mixed pentamethine oxonols preparedfrom 1 equivalent of the barbiturate with functionalized linkers and1,3-dibutyl thiobarbiturate have been characterized. An exemplarysynthesis is depicted in FIG. 11. It will be recognized that oxonolswith alkyl chains of length other than C₅ can be readily prepared bysuch a method and are contemplated as within the scope of thisinvention.

[0208] One preferred class of suitable linkers includesbi-functionalized polyalkylene glycol oligomers (polyethyleneglycol,polypropyleneglycol, polybutyleneglycol, etc.) of an appropriate lengthto span the plasma membrane (25-30 carbon equivalents), for example 8-10PEG units. The oxygen (or sulfur, in the corresponding thio-analogsthereof) modulates the hydrophobicity and hence translocation rate andloading. Compounds joined by such linker groups have the general formula

X—(CH₂)_(m)—Z_(q)—(CH₂)_(m′)—Z′_(q′)—(CH₂)_(m″)—Z″_(q″)—Y

[0209] wherein:

[0210] X is a hydrophobic fluorescent anion;

[0211] Y is a fluorescent second reagent;

[0212] Z, Z′, Z″ are independently O, S, SS, CO, COO;

[0213] m, m′ and m″ are integers from 0 to about 32;

[0214] q, q′, and q″ are independently 0 or 1; and

[0215] m+q+m′+q′+m″+q″ is from about 20 to 40 (preferably between 25 and35).

[0216] Preferably Z is S, i.e., the linkers are polyalkylene thioethers;m=5, Z−S, q=1, m′=12, Z′=S, q′=1, m″=11, Z″=CO, and q″=1.

[0217] Another class of suitable linkers includes functionalized alkylchains with terminal thiol groups that form a central disulfide linkage.The disulfide functions as a hydrophobic swivel in the center of themembrane. These compounds have the general formula

X—(CH₂)_(n)SS(CH₂)2_(n′)—Y

[0218] wherein:

[0219] X is a hydrophobic fluorescent anion;

[0220] Y is a fluorescent second reagent;

[0221] n and n′ are integers from 0 to about 32 wherein n+n′ is lessthan or equal to 33.

[0222] As would be readily appreciated by those skilled in the art, thelinker groups may be reacted with appropriately substituted orfunctionalized first and second fluorophores using conventional couplingchemistries. Further, it is evident that the linker group may beattached to a fluorophore at a variety of different positions. Importantlocations (X) for attachment of the linker in exemplary classes ofoxonols are illustrated in FIG. 12.

IV. MEASUREMENT METHODS

[0223] In one class of embodiments of the present invention, thehydrophobic ion fluorescence on one face of the membrane is quenched bya mechanism other than FRET. FRET has the advantages of working overlong distances, which minimizes the necessary concentration ofacceptors, and of giving ratiometric output at two emission wavelengths.However, if FRET is too efficient over very long distances greater thanthe thickness of the membrane, it can fail to discriminate betweenacceptors on the same vs. opposite sides of the membrane. The othermechanisms of quenching are much shorter-range and should never beeffective across the thickness of the membrane.

[0224] The second fluorophore/quencher can be located on either theintracellular or the extracellular face, as long as it is specific toone or the other. In the specific examples reported herein, theextracellular surface was targeted for convenience.

[0225] FRET or fluorescence quenching is best detected by emissionratioing which can distinguish the two populations of the mobilefluorophore, i.e, those bound to the extracellular vs. those bound tothe intracellular face of the membrane. In particular, FRET using afluorescent acceptor provide an emission ratio change that is wellsuited to laser-scanning confocal microscopy and internally corrects forvariations in donor loading, cell thickness and position (includingmotion artifacts), and excitation intensity. Emission ratios usuallychange by larger percentages than either emission wavelength signalalone, because the donor and acceptor emissions should change inopposite directions, which reinforce each other when ratioed. Ifemission ratioing is not desirable or possible, either wavelength canstill be used alone, or the change in donor excited-state lifetimemonitored.

[0226] Emission ratios are measured either by changing the passband of awavelength-selective filter in front of a single detector, or preferablyby splitting the emitted light with a dichroic mirror and measuring twowavelength bands simultaneously with two detectors, which may each bepreceded by additional wavelength-selecting filters. In the firstmethod, the wavelength-selective filters may be two or more interferencefilters with different passbands alternately placed in front of thedetector, or they may be a continuously tunable monochromator which isrepeatedly scanned over a wavelength range. The advantage of the firstmethod is that only one detector is used, which economizes on detectorsand avoids the problem of precisely matching two detectors. Theadvantages of the second method, using a dichroic mirror and twoseparate detectors, are that the two emissions may be measured trulysimultaneously rather than sequentially, and that it makes moreefficient use of the photons emitted from the sample.

[0227] Molecular specificity for particular cell types in a mixedpopulation may be introduced by using cell-specific antibodies orlectins as the carriers of the extracellular fluorescent label, or byusing Green Fluorescent Protein specifically expressed in a given celltype as the intra- or extracellular label. Specifically labeled cellsalso reduce background staining and provide large fluorescence changesin complex tissue.

[0228] High sensitivity is achieved when the voltage sensor (i.e., thehydrophobic anion of the first reagent) translocates at least a fullunit charge nearly all the way through the membrane. Even withoutspecific ion channels or transporters, such translocation can be quiterapid if the ion is negatively charged, delocalized, and hydrophobic.For example, the lipid-soluble non-fluorescent anion of dipicrylamine(2,2′,4,4′,6,6′-hexanitrodiphenylamine) produces displacement currentsin excitable tissue with submillisecond kinetics, comparable in speed tosodium channel gating currents [Benz, R. and Conti, F. 1981. Structureof the squid axon membrane as derived from charge-pulse relaxationstudies in the presence of absorbed lipophilic ions. J. Membrane Biol.59:91-104; Benz, R. and Nonner, W. 1981. Structure of the axolemma offrog myelinated nerve: relaxation experiments with a lipophilic probeion. J. Membrane Biol. 59:127-134; Fernández, J. M., Taylor, R. E., andBezanilla, F. 1983. Induced capacitance in the squid giant axon. J. Gen.Physiol. 82:331-346]. However, voltage sensing should not requirefurther diffusion of the ion through the unstirred aqueous layers,because that slows the response and generates a sustained leakagecurrent.

[0229] To create an optical readout from the translocation of thefluorescent hydrophobic ion (i.e., the first reagent) from one side ofthe plasma membrane to the other side, FRET or fluorescence quenchingbetween the translocating ion and a fluorophore or quencher (i.e., thesecond reagent) fixed to just one face of the plasma membrane isemployed. Most conveniently, the extracellular face is employed.

[0230] By way of example, and not limitation, the case where thetranslocating ions are anionic fluorescent acceptors which absorb atwavelengths that overlap with the emission spectrum of theextracellularly fixed donor fluorophores is schematically shown inFIG. 1. At a resting negative membrane potential (A) permeable oxonolshave a high concentration at the extracellular surface of the plasmamembrane and energy transfer from the extracellularly bound FL-WGA(fluorescein-wheat germ agglutinin) is favored. FRET is symbolized bythe straight arrow from lectin to oxonol. At a positive membranepotential (B) the anions are located primarily on the intracellularsurface of the membrane and energy transfer is greatly reduced becauseof their increased mean distance from the donors on the extracellularsurface.

[0231] The speed of the voltage-sensitive fluorescence response dependson the translocation rate of the fluorophore from one site to-the other.The speed of response for DiSBA-C₆-(3) is shown in FIG. 5 and followsthe general equations (1) and (2). As this equation indicates,fluorescent ions which jump across the membrane on a millisecondtimescale in response to biologically significant changes intransmembrane potential are needed to follow rapidpolarization/depolorization kinetics. Slower-jumping ions would not beuseful, for example, in following fast electrical signals in neuronaltissue (a primary application of the compositions and methods of thepresent invention). The development and discovery of such molecules withthe added constraint of being fluorescent is not trivial.

[0232] The mobile hydrophobic anions can be donors rather thanacceptors. Each of the alternatives has its own advantages. An examplewith the hydrophobic ion being the FRET donor is the DiSBA-C₆-(3)/TexasRed WGA combination. A primary advantage of this arrangement is that itminimizes the concentration of the hydrophobic dye molecule in themembrane; this reduces toxicity and cellular perturbations resultingfrom the displacement current and any photodynamic effects. Anotheradvantage is the generally higher quantum yields of fluorophores boundin membranes relative to those on proteins or water; this gives betterFRET at a given distance.

[0233] Bis-(1,3-dialkyl-2-thiobarbiturate)-trimethineoxonols, wherealkyl is n-hexyl and n-decyl (DiSBA-c₆-(3) and DiSBA-C₁₀-(3)respectively) have been shown herein to function as donors to Texas Redlabeled wheat germ agglutinin (TR-WGA) and as acceptors from fluoresceinlabeled lectin (FL-WGA). In voltage-clamped fibroblasts, thetranslocation of these oxonols was measured as a displacement currentwith a time constant of about 2 ms for 100 mV depolarization at 20° C.,which equals the speed of the fluorescence changes. Fluorescence ratiochanges of between 4-34% were observed for a 100 mV depolarization infibroblasts, astrocytoma cells, beating cardiac myocytes, and B104neuroblastoma cells. The large fluorescence changes allowed high speedconfocal imaging.

[0234] Single cells were used in the examples so that the opticalsignals could be compared with voltage changes accurately known fromtraditional microelectrode techniques, such as patch clamping, which areapplicable only to single cells. However, it should be apparent that thedyes can be used for many applications in which microelectrodes are notapplicable. Comparison with microelectrodes is needed merely foraccurate calibration and proof that the mechanism of fluorescence signalgeneration is as described herein. The two reagent compositions andmethods described herein can either resolve the different electricalpotentials of many neighboring cells or neighboring parts of a singlecell, or give an average reading for all the membrane locations,depending on whether the optical signal is spatially imaged or pooled.

[0235] The methods described herein are applicable to a wide variety ofmembranes. In particular, membrane potentials in membranes of biologicalcells can be detected and monitored. The method finds greatest utilitywith plasma membranes, especially the outermost plasma membrane ofmammalian cells. Representative membranes include, but are not limitedto, subcellular organelles, membranes of the endoplasmic reticulum,secretory granules, mitochondria, microsomes and secretory vesicles.Cell types which can be used include but are not limited to, neurons,cardiac cells, lymphocytes (T and B lymphocytes, nerve cells, musclecells and the like.

V. DRUG SCREENING

[0236] The invention also provides methods for screening test samplessuch as potential therapeutic drugs which affect membrane potentials inbiological cells. These methods involve measuring membrane potentials asdescribed above in the presence and absence (control measurement) of thetest sample. Control measurements are usually performed with a samplecontaining all components of the test sample except for the putativedrug. Detection of a change in membrane potential in the presence of thetest agent relative to the control indicates that the test agent isactive. Membrane potentials can be also be determined in the presence orabsence of a pharmacologic agent of known activity (i.e., a standardagent) or putative activity (i.e., a test agent). A difference inmembrane potentials as detected by the methods disclosed herein allowsone to compare the activity of the test agent to that of the standardagent. It will be recognized that many combinations and permutations ofdrug screening protocols are known to one of skill in the art and theymay be readily adapted to use with the method of membrane potentialmeasurement disclosed herein to identify compounds which affect membranepotentials. Use of the membrane potential determination techniquedisclosed herein in combination with all such methods are contemplatedby this invention. In a particular application, the invention offers amethod of identifying a compound which modulates activity of an ionchannel, pump, or exchanger in a membrane, comprising:

[0237] (a) loading the cells with the first and second reagents, whichtogether measure membrane potential as described above;

[0238] (b) determining the membrane potential as described above;

[0239] (c) exposing the cells to the test sample;

[0240] (d) redetermining the membrane potential and comparing with theresult in (b) to determine the effect of the test sample;

[0241] (e) optionally, exposing the membrane to a stimulus whichmodulates an ion channel, pump or exchanger, and redetermining themembrane potential and comparing with the result in (d) to determine theeffect of the test sample on the response to the stimulus.

[0242] In another application, the invention offers a method ofscreening test samples to identify a compound which modulates theactivity of an ion channel, pump or exchanger in a membrane, comprising:

[0243] (a) loading a first set and a second set of cells with first andsecond reagents which together measure membrane potential;

[0244] (b) optionally, exposing both the first and second set of cellsto a stimulus which modulates the ion channel, pump or exchanger;

[0245] (c) exposing the first set of cells to the test sample;

[0246] (d) measuring the membrane potential in the first and second setsof cells; and

[0247] (e) relating the difference in membrane potentials between thefirst and second sets of cells to the ability of a compound in the testsample to modulate the activity of an ion channel, pump or exchanger ina membrane.

[0248] Ion channels of interest include, but are not limited to, sodium,calcium, potassium, nonspecific cation, and chloride ion channels, eachof which may be constitutively open, voltage-gated, ligand-gated, orcontrolled by intracellular signaling pathways.

[0249] Biological cells which can be screened include, but are notlimited to primary cultures of mammalian cells, cells dissociated frommammalian tissue, either immediately or after primary culture. Celltypes include, but are not limited to white blood cells (e.g.leukocytes), hepatocytes, pancreatic beta-cells, neurons, smooth musclecells, intestinal epithelial cells, cardiac myocytes, glial cells, andthe like. The invention also includes the use of recombinant cells intowhich ion transporters, ion channels, pumps and exchangers have beeninserted and expressed by genetic engineering Many cDNA sequences forsuch transporters have been cloned (see U.S. Pat. No. 5,380,836 for acloned sodium channel) and methods for their expression in cell lines ofinterest is within the knowledge of one of skill in the art (see, U.S.Pat. No. 5,436,128). Representative cultured cell lines derived fromhumans and other mammals include LM (TK⁻) cells, HEK293 (human embryonickidney cells), 3T3 fibroblasts, COS cells, CHO cells, RAT1 and HLHepG2cells.

[0250] The screening methods described herein can be made on cellsgrowing in or deposited on solid surfaces. A common technique is to usea microtiter plate well wherein the fluorescence measurements are madeby commercially available fluorescent plate readers. The inventionincludes high throughput screening in both automated and semiautomatedsystems. One such method is to use cells in Costar 96 well microtiterplates (flat with a clear bottom) and measure fluorescent signal withCytoFluor multiwell plate reader (Perseptive Biosystems, Inc., MA) usingtwo emission wavelengths to record fluorescent emission ratios.

[0251] The invention may be better understood with reference to theaccompanying examples, which are intended for purposes of illustrationonly and should not be construed as in any sense limiting the scope ofthe invention as defined in the claims appended hereto.

EXAMPLES Example I

[0252] Synthesis of Oxonol Dyes

[0253] All starting materials and reagents were of the highest purityavailable (Aldrich; Milwaukee, Wis.) and used without furtherpurification, except where noted. Solvents were HPLC grade (Fisher) andwere dried over activated molecular sieves 3Å. NMR spectra were acquiredon a Varian Gemini 200 MHz spectrometer (Palo Alto, Calif.). The spectrawere referenced relative to the residual solvent peak (CHCl₃,=7.24 ppm).Fluorescence spectra were taken on a SPEX Fluorolog-2 (Edison, N.J.) andwere corrected for lamp and detector wavelength variations using themanufacturer supplied correction files.

[0254] Bis-(1,3-dibutyl-2-thiobarbiturate)-trimethineoxonolDiSBA-C₄-(3):

[0255] DiSBA-C₄-(3) was synthesized based on the procedure for the ethylderivative [British Patent 1,231,884]. 1,3-di-butyl-thiobarbiturate (500mg, 2 mmol) was dissolved in 700 μL of pyridine. To this solution, amixture of 181 μL (1.1 mmol) of malonaldehyde bis(dimethyl acetal) and100 μL of 1 M HCl was added. The solution immediately turned red. After3 h, half of the reaction mixture was removed and 2 equiv. of theprotected malonaldehyde was added every hour for 4 h to the remainingmixture. The reaction mixture was then filtered to collect purple/blackcrystals of the DiSBA-C₄-(3) pyridinium salt. After washing the crystalswith water and then drying under vacuum (0.5 torr), 67.2 mg of pureproduct was collected. ¹H NMR (CDCl₃): 8.91 (2H, d, J=5.1 Hz, py), 8.76(1H, t, J=13.7 Hz, central methine), 8.52 (1H, t, J=8.0 Hz, py), 8.16(2H, d, J=13.9 Hz, methine), 8.00 (2H, dd, J₁-J₂=6.9 Hz, py), 4.47 (8H,cm, NCH ₂CH₂CH₂CH₃) , 1.69 (8H, cm, NCH₂CH ₂CH₂CH₃), 1.39 (8H, cm,NCH₂CH₂CH ₂CH₃), 0.95 (12H, t, J=6.4 Hz, methyl).

[0256] To prepare 1,3-di-butyl-thiobarbiturate, 1.22 g of Na (53 mmol)was slowly dissolved in 20 mL of dry ethanol under argon. To theethoxide solution, 8.5 g (8 mL, 53 mmol) of diethyl malonate followed by5 g (26.5 mmol) of dibutylthiourea were added. The reaction mixture washeated and refluxed for 3 days. After cooling, the mixture was filtered.The filtrate was clarified with addition of water. Concentrated HCl wasthen added until the pH was 1-2. The acidic filtrate was then extracted3× with hexanes. The extract was concentrated and 5.5 g of crude productprecipitated out of solution. The solid was recrystallized from methanolwith addition of small amounts of water yielding 4.23 g of the purebarbituric acid (65%). ¹H NMR (CDCl₃): 4.33 (4H, cm, NCH ₂CH₂CH₂CH₃) ,3.71 (2H, s, ring CH₂) , 1.63 (4H, cm, NCH₂CH ₂CH₂CH₃) 1.35 (4H, cm,NCH2CH₂CH ₂CH₃) 0.94 (6H, t, J=6.2 Hz, methyl).

[0257] Bis-(1,3-dihexyl -2-thiobarbiturate)-pentamethineoxonol(DiSBA-C₆-(5)):1,3-dihexyl-2-thiobarbituric acid (200 mg, 0.64 mmol) andglutacondialdehyde dianil monohydrochloride (whose Chem. Abs. name isN-[5-(phenylamino)-2,4-pentadienylidene]benzenamine, monohydrochloride)(91 mg, 0.32 mmol) were mixed in 1 mL pyridine. Within 10 s, thesolution turned blue. After letting the reaction mixture stir for 1.5 h,the solvent was removed under high vacuum. The residue was dissolved inCHCl₃ and chromatographed on silica gel eluting with a (93:7) CHCl₃/MeOHsolution. The pure blue oxonol (72 mg) was recovered. ¹HNMR(CDCl₃/CD₃OD): d 7.60-7.80 (cm, 4H, methines), 7.35 (t, J=11.3 Hz, 1H,central methine), 4.31 (cm, 8H, NCH₂R), 1.57 (cm, 8H, NCH₂CH₂R), 1.20(br m, 24H, bulk methylenes), 0.74 (br t, 12H, methyl).

[0258] Other oxonols were made using the same procedure, starting withthe appropriate thiourea prepared from requisite primary amine andcarbon disulfide [Bortnick, N., Luskin, L. S., Hurwitz, M. D., andRytina, A. W. 1956. t-Carbinamines, RR′R″CNH₂. III. The preparation ofisocyanates, isothiocyanates and related compounds. J. Am. Chem. Soc.78:4358-4361]. An exemplary synthesis of Di-SBA-C₆-(3) is depicted inFIG. 13.

Example II

[0259] Synthesis of Flourescent Phospholipids

[0260] Cou-PE:3-amidoglycine-6-chloro-7-butyryloxy coumarin wassynthesized as described in pending U.S. patent application, Ser. No.08/407,554, filed Mar. 20, 1995 as set out below. For synthesis of 2,4dihydroxy-5-chlorobenzaldehyde, 21.7 g (0.15 Mol) 4-chlororesorcinolwere dissolved in 150 ml dry diethyl ether and 27 g finely powdered zinc(II) cyanide and 0.5 g potassium chloride were added with stirring. Thesuspension was cooled on ice. A strong stream of hydrogen chloride gaswas blown into the solution with vigorous stirring. After approximately30 minutes the reactants were dissolved. The addition of hydrogenchloride gas was continued until it stopped being absorbed in the ethersolution (approx. 1 hour). During this time a precipitate formed. Thesuspension was stirred for one additional hour on ice. Then the solidwas let to settle. The ethereal solution was poured from the solid. Thesolid was treated with 100 g of ice and heated to 100 degrees C. in awater bath. Upon cooling the product crystallized in shiny plates fromthe solution. They were removed by filtration on dried over potassiumhydroxide. The yield was 15.9 g (0.092 Mol, 61%). ¹H NMR (CDCl₃): δ 6.23ppm (s, 1H, phenol), δ 6.62 ppm (s, 1H, phenyl), δ 7.52 ppm (s, 1H,phenyl), δ 9.69 ppm (s, 1H, formyl), δ 11.25 ppm (s, 1H, phenol).

[0261] To prepare 3-carboxy 6-chloro 7-hydroxy coumarin, 5.76 g (0.033Mol) 2,4-dihydroxy-5-chlorobenzaldehyde and 7.2 g (0.069 Mol) malonicacid were dissolved in 5 ml warm pyridine. 75 microliters aniline werestirred into the solution and the reaction let to stand at roomtemperature for 3 days. The yellow solid that formed was broken intosmaller pieces and 50 ml ethanol was added. The creamy suspension wasfiltered through a glass frit and the solid was washed three times with1 N hydrochloric acid and then with water. Then the solid was stirredwith 100 ml ethyl acetate, 150 ml ethanol and 10 ml half concentratedhydrochloric acid. The solvent volume was reduced in vacuo and theprecipitate recovered by filtration, washed with diethyl ether and driedover phosphorous pentoxide. 4.97 g (0.021 Mol, 63%) of product wasobtained as a white powder. 1H NMR (dDMSO): δ 6.95 ppm (s, 1H), δ 8.02ppm (s, 1H), δ 8.67 ppm (s, 1H).

[0262] To prepare 7-butyryloxy-3-carboxy-6-chlorocoumarin, 3.1 g (12.9mMol) 3-carboxy-6-chloro-7-hydroxycoumarin were dissolved in 100 mldioxane and treated with 5 ml butyric anhydride, 8 ml pyridine and 20 mgdimethyl aminopyridine at room temperature for two hours. The reactionsolution was added with stirring to 300 ml heptane upon which a whiteprecipitate formed. It was recovered by filtration and dissolved in 150ml ethyl acetate. Undissolved material was removed by filtration and thefiltrate extracted twice with 50 ml 1 N hydrochloric acid/brine (1:1)and then brine. The solution was dried over anhydrous sodium sulfate.Evaporation in vacuo yielded 2.63 g (8.47 mMol, 66%) of product. ¹H NMR(CDCl₃): δ 1.08 ppm (t, 3H, J=7.4 Hz, butyric methyl), δ 1.85 ppm (m,2H, J₁≈J₂=7.4 Hz, butyric methylene), δ 2.68 ppm (t, 2H, J=7.4 Hz,butyric methylene), δ 7.37 ppm (s, 1H, coumarin), δ 7.84 ppm (s, 1H,coumarin), δ 8.86 ppm (s, 1H, coumarin).

[0263] Preparation of7-butyryloxy-3-benzyloxycarbonylmethylaminocarbonyl-6-chlorocoumarin iseffected as follows. 2.5 g (8.06 mMol)7-Butyryloxy-3-carboxy-6-chlorocoumarin, 2.36 g hydroxybenztriazolehydrate (16 mMol) and 1.67 g (8.1 mMol) dicyclohexyl carbodiimide weredissolved in 30 ml dioxane. A toluene solution of O-benzylglycine[prepared by extraction of 3.4 g (10 mMol) benzylglycine tosyl salt withethyl acetate-toluene-saturated aqueous bicarbonate-water (1:1:1:1, 250ml), drying of the organic phase with anhydrous sodium sulfate andreduction of the solvent volume to 5 ml] was added dropwise to thecoumarin solution. The reaction was kept at room temperature for 20hours after which the precipitate was removed by filtration and washedextensively with ethylacetate and acetone. The combined solventfractions were reduced to 50 ml on the rotatory evaporator upon whichone volume of toluene was added and the volume further reduced to 30 ml.The precipitating product was recovered by filtration and dissolved in200 ml chloroform—absolute ethanol (1:1). The solution was reduced to 50ml on the rotatory evaporator and the product filtered off and dried invacuo yielding 1.29 g of the title product. Further reduction of thesolvent volume yielded a second crop (0.64 g). Total yield: 1.93 g (4.22mMol, 52%). ¹H NMR (CDCl₃): δ 1.08 ppm (t, 3H, J=7.4 Hz, butyricmethyl), δ 1.84 ppm (m, 2H, J₁≈J₂=7.4 Hz, butyric methylene), δ 2.66 ppm(t, 2H, J=7.4 Hz, butyric methylene), δ 4.29 ppm (d, 2H, J=5.5 Hz,glycine methylene), δ 5.24 ppm (s, 2H, benzyl), δ 7.36 ppm (s, 1H,coumarin), δ 7.38 ppm (s, 5H, phenyl), δ 7.77 ppm (s, 1H, coumarin), δ8.83 ppm (s, 1H, coumarin), δ 9.15 ppm (t, 1H, J=5.5 Hz, amide).

[0264] 7-Butyryloxy-3-carboxymethylaminocarbonyl-6-chlorocoumarin wasprepared as follows. 920 mg (2 mMol)7-butyryloxy-3-benzyloxycarbonylmethylaminocarbonyl-6-chlorocoumarinwere dissolved in 50 ml dioxane. 100 mg palladium on carbon (10%) and100 microliters acetic acid were added to the solution and thesuspension stirred vigorously in a hydrogen atmosphere at ambientpressure. After the uptake of hydrogen seized the suspension wasfiltered. The product containing carbon was extracted five times with 25ml boiling dioxane. The combined dioxane solutions were let to cool uponwhich the product precipitated as a white powder. Reduction of thesolvent to 20 ml precipitates more product. The remaining dioxanesolution is heated to boiling and heptane is added until the solutionbecomes cloudy. The weights of the dried powders were 245 mg, 389 mg and58 mg, totaling 692 mg (1.88 mMol, 94%) of white product. ¹H NMR(dDMSO): δ 1.02 ppm (t, 3H, J=7.4 Hz, butyric methyl), δ 1.73 ppm (m,2H, J₁≈J₂=7.3 Hz, butyric methylene), δ 2.70 ppm (t, 2H, J=7.2 Hz,butyric methylene), δ 4.07 ppm (d, 2H, J=5.6 Hz, glycine methylene), δ7.67 ppm (s, 1H, coumarin), δ 8.35 ppm (s, 1H, coumarin), δ 8.90 ppm (s,1H, coumarin), δ 9.00 ppm (t, 1H, J=5.6 Hz, amide).

[0265] 6-chloro-7-(n-butyryloxy)coumarin-3-carboxamidoacetic acid (26.2mg, 100 mmol) was dissolved in 2 mL of 1:1 CHCl₃/dioxane.Isobutylchloroformate (14.3 mL, 110 mmol) was added under Ar at 40° C.and left stirring for 30 min. Separately,dimyristoylphosphatidylethanolamine (DMPE) (20 mg, 31.5 mmol) wasdissolved in 1 mL of CHCl₃ with 1 drop of dry MeOH and 6 mL (34.5 mmol)of diisopropylethylamine (DIEA) added. The mixed anhydride solution wasthen pipetted into the phospholipid solution. After 2 h, the solvent wasremoved under vacuum. The residue was dissolved in 3 mL of MeOH andmixed with 3 mL of 0.25 M NaHCO₃. The solution almost immediately turnyellow and was stirred for 15 min. The solution was then extracted 3-5times with CHCl₃. A bad emulsion is formed. The extracts were combinedand concentrated. The residue was dissolved in 1 mL 1:1 MeOH/H₂O andpurified on a C₁₈ reverse phase column (1.7×7 cm). Eluting with the samesolvent, a fluorescent band passed through the column, followed by aslower one. The solvent polarity was decreased to 9:1 MeOH/H₂O and themajor yellow band then eluted off the column. After concentration anddrying 2.5 mg (2.74 mmol) of pure product was collected. ¹HNMR ( CD₃OD):d 8.72 (s, 1H, coumarin), 7.81 (s, 1 H, coumarin), 6.84 (s, 1H,coumarin), 5.25 (cm, 2H), 4.43 (dd, J₁=12.1 Hz, J₂=3.2 Hz), 4.22 (d,J=6.6 Hz, 1 H), 4.13 (s, ⁻4H), 3.7-4.1 (cm, ⁻11 H), 3.47 (cm, ⁻3H),3.2-3.3 (⁻q), 2.31 (cm ⁻7H), 1.57 (br s, ⁻8H, CH₂ a to carbonyl),1.2-1.5 (cm, ⁻63H, bulk CH₂′s), 0.92 (unres t, ⁻12H, CH₃). Electrospray(neg. ion) MS [MeOH/H_(2l O:) 95/5] (peak, rel. int.) 456.8 (M⁻², 20),524.5 (50), 704.9 (6), 734.7 (100), 913.9 (M⁻¹, 95); deconvolutedM=915.3 amu; calc. M=915.5 amu. UV-vis (MeOH/HBSS; 2/1) 1_(max)=414 nm.Fluorescence (MeOH/HBSS; 2/1) 1_(emax)=450 nm, Quantum Yield=b 1.0.

[0266] Cy5-PE:DMPE (1.0 mg, 1.6 mmol) was dissolved in 650 mL of (12:1)CHCl₃/MeOH and DIEA (1 mL, 5.7 mmol) was added. Separately, Cy5-OSu(Amersham; Arlington Heights, Ill.), the N-hydroxysuccinimide ester ofN-ethyl-N′-(5-carboxypentyl)-5,5′-disulfoindodicarbocyanine, (0.8 mg, 1mmol) was dissolved in 150 mL of (2:1) CHCl₃/MeOH and added to thephospholipid solution. After 3 h, the solvent was removed under vacuum.The residue was dissolved in MeOH and loaded on a C₁₈ reverse phasecolumn (1×10 cm) equilibrated with 1:1 MeOH/H₂O. Eluting with the samesolvent, the hydrolyzed ester was removed. The polarity was decreased to9:1 MeOH/H₂O and the pure blue product was eluted off the column,yielding 400 mg (310 nmol, 31%).

Example III

[0267] Synthesis of Linker for Donors and Acceptors

[0268] This example is with reference to FIGS. 14-16.12-p-methoxybenzylthio-1-dodecanol (1): Na (800 mg, 34.8 mmol) wasdissolved in 30 mL of dry MeOH. Under argon, p-methoxybenzylmercaptan(2.75 mL, 3.04 g, 19.7 mmol) was added to the methoxide solution. Aftera few minutes, 12-bromododecanol (2.5 g, 9.43 mmol) was dropped into thereaction mixture. Within 5 minutes a solid began to come out ofsolution. After a minimum of 3 h, the reaction was filtered and washed3× with cold MeOH, yielding 2.874 g (8.49 mmol, 90%) of pure productafte- drying. ¹H NMR (CDCl₃): d 7.23 (d, J=8.8 Hz, 2H, AA′ of AA′ BB′aromatic system), 6.85 (d, J=8.8 Hz, 2H, BB′ of AA′ BB′ aromaticsystem), 3.80 (s, 3H, methoxy), 3.66 (s, 2H, benzyl), 3.64 (dt, J₁=6.6Hz, J₂=5.5 Hz, 2H, RCH₂OH), 2.40 (t, J=7.3 Hz, 2H, RSCH₂R), 1.50-1.65(cm, 4H, CH₂ b to heteroatoms), 1.2-1.4 (cm, 16H, bulk methylenes).

[0269] 12-p-methoxybenzylthio-1-bromododecane (2): (1) (500 mg, 1.48mmol) was mixed with carbon tetrabromide (611 mg, 1.85 mmol) in 2.5 mLCH₂Cl₂ and cooled in an ice bath until solid began to come out ofsolution. The ice bath was removed and triphenylphosphine (348 mg, 2.22mmol) was added to the reaction. The solution immediately turnedyellowish. The starting material had been consumed after 30 minaccording to TLC (EtOAc/Hex, 1:1). The solvent was removed and 50 mL ofhexane was added to the solid residue. After stirring overnight, thesolution was filtered and concentrated to a solid. The solid was thenmixed with about 10-15 mL of hexane and again filtered. The concentratedfiltrate yielded 537 mg (1.34 mmol, 91%) of pure product after drying.¹H NMR (CDCl₃): d 7.23 (d, J=8.6 Hz, 2H, AA′ of AA′ BB′ aromaticsystem), 6.85 (d, J=8.7 Hz, 2H, BB′ of AA′ BB′ aromatic system), 3.80(s, 3H, methoxy), 3.67 (s, 2H, benzyl), 3.41 (t, J=6.9 Hz, 2H, RCH₂Br),2.40 (t, J=7.3 Hz, 2H, RSCH₂R), 1.86 (cm, 2H, CH₂ b to Br), 1.15-1.45(cm, 18H, bulk methylenes).

[0270] 12-(12-p-methoxybenzylthio-1-dodecylthio)-dodecanoic acid (3): Na(116 mg, 5 mmol) was dissolved it dry MeOH. 12-mercapto-1-dodecanoicacid (340 mg, 1.46 mmol)—synthesized according to JACS 115, 3458-3474,1993—was added to the methoxide solution. After stirring with someheating for 5 min, (2) (497 mg, 1.24 mmol) was added to the reaction.The reaction became very viscous and an additional 1.75 mL of MeOH wasintroduced. The reaction was then left overnight. The reaction wasquenched with 10% acetic acid. The paste-like reaction mixture wastransferred to a 500 mL separatory funnel dissolved in equal volumes ofEtOAc/Hex (1:1) and the acetic acid solution. The organic layer wasseparated. The aqueous layer was then extracted two more times. Thecombine extracts were concentrated yielding 740.3 mg (1.34 mmol) ofcrude product. The excess acetic acid was removed as a tolueneazeotrope. The solid was crystallized from isopropyl ether giving 483 mg(71%). TLC and NMR show an impurity believed to be a disulfide sideproduct. The material was further purified by flash chromatographyeluting with CHCl₃/ MeOH/AA (99:0.5:0.5) yielding 334 mg (0.604 mmol,49%) of pure product. ¹H NMR (CDCl₃): d 9.45 (brs, 1H, COOH), 7.23 (d,J=8.8 Hz, 2H, AA′ of AA′ BB′ aromatic system), 6.85 (d, J=8.7 Hz, 2H,BB′ of AA′ BB′ aromatic system), 3.80 (s, 3H, methoxy), 3.66 (s, 2H,benzyl), 2.50 (t, J=7.3 Hz, 4H, RCH₂SCH₂R) , 2.40 (t, J=7.3 Hz, 2H,RSCH₂R) , 2.35 (t, J=7.5 Hz, 2H, RCH₂COOH), 1.5-1.7 (cm, 8H, CH₂ b toheteroatoms), 1.15-1.45 (cm, 30H, bulk methylenes). ¹³C NMR (CDCl₃): d179.5 (COOH), 129.9 (aromatic, 2C), 113.9 (aromatic, 2C), 55.2 (MeOR) ,35.6 (CH₂), 33 .7 (CH₂), 32.2 (CH₂) , 31.3 (CH₂), 29.7 (CH₂) , 29.4(CH₂), 29.2 (CH₂) , 28.9 (CH₂), 24.6 (CH₂).

[0271] 1-butyl-3-(12-(12-pentylthio-1-dodecylthio)-dodecanoic acid)thiobarbiturate (13): (3) (73.8 mg, 133.5 μmol) was deprotected in 2 mLof dry TFA/anisole (14:1) at 70° C. for 2.5 h. The solvent was removedunder vacuum and the residue was dissolved in 10 mL dry EtOH. Sodiumborohydride (330 mg) was added and the mixture was stirred overnight.The solution was then acidified with conc. HCL until gas stoppedevolving. The solution was then extracted with ether 4×. The combinedextracts were concentrated leaving a white solid. The solid was thendissolved and concentrated 2× with degassed MeOH. After drying on thehigh vacuum, 69.5 mg of solid was recovered. It was estimated by TLCthat this solid was 1:1 the deprotected product anddi-p-methoxyphenylmethane, (104 μmol, 78%). The deprotected linker wasdissolved in 0.5 mL dry DMF, with heating. NaH (⁻550 μmol) was addedwhich caused some gas evolution. (8) (38.5 mg, ⁻100 μmol) was then addedto the reaction in 100 uL DMF and the reaction was left overnight at 60°C. TLC in EtOAc/MeOH/AA (90:8:2) indicated that a new more non-polarbarbituric acid had been formed. The solvent was removed and the residuewas dissolved in EtOAc/Hex (1:1) and washed with water. The material wasthen purified by chromatography, eluting with EtOAc/MeOH/AA (90:8:2).NMR of the product showed resonance from barbituric acid and the linker.Electrospray (neg. ion) MS [MeOH/H₂O: 95/5] (peak, rel. int.) 516.4(95), 699.4 (M⁻¹, 100), 715.1 (M⁻¹+16, 40), 1024.0 (M⁻¹+32, 25); calc.M⁻¹=700.1 amu. The ether linkers appears to be partially oxidized tosulfoxides.

[0272] 1-(1,3-dibutylthiobarbiturate)-3-(1-butyl-3-(12-(12-pentylthio-1-dodecylthio)-dodecanoicacid) thiobarbiturate) trimethineoxonol (14): (3) (73.8 mg, 133.5 mol)was deprotected in 2 mL of dry TFA/anisole (14:1) at 70° C. for 2.5 h.The solvent was removed under vacuum and the residue was dissolved in 20mL dry EtOH. Sodium borohydride (330 mg) was added and the mixture wasstirred overnight. The solution was then acidified with conc. HCL untilgas stopped evolving. The solution was then extracted with ether 4×. Thecombined extracts were concentrated leaving a white solid. The solid wasthen dissolved and concentrated 2× with degassed MeOH. After drying onthe high vacuum, 71.1 mg of solid was recovered. It was estimated by TLCthat this solid was 1:1 the deprotected product anddi-p-methoxyphenylmethane, (106 μmol, 79%). The deprotected linker wasdissolved in 1 mL dry DMF, with heating. NaH (⁻350 μmol) was added whichcaused some gas evolution. (12) (35.5 μmol) was then added to thereaction in 200 uL DMF. The reaction did not seem to be proceeding after1 h, so 4 mg of N.H. (60%) was added to the reaction mixture. Thesolution now appeared orange instead of red and a second non-polaroxonol began to form. The reaction, heated at 60° C., was allowed to gofor 18 h. Half of the reaction mixture was worked up as follows. Thereaction mixture was transferred to a 30 mL separatory funnel in ⁻12 mLtoluene. About 4 mL a 10% acetic acid solution and 3 mL of water wereadded. Most of the oxonol partitioned into the organic layer, which waswashed 3× with acetic acid solution/water (1:1). The organic layer wasthen concentrated and purified by flash chromatography (2.5×18 cm). Thecolumn was packed and first eluted with CHCl₃/MeOH/AA (93:5:2)- After 1non-polar oxonol was removed, the solvent polarity was increased toCHCl₃/MeOH/AA (90:8:2). This caused the oxonol product to elute off thecolumn. After concentrating the fractions and drying, 7.2 mg pureproduct (7.25 μmol, 20%) was attained. ¹H NMR ( CDCl₃/MeOH): d 8.54 (t,J=13.8 Hz, 1H, central methine), 7.97 (d, J=14.2 Hz, 2H, methines), 4.39(cm, 8H, NCH₂R), 2.46 (t, J=7.3 Hz, 8H, RCH₂SCH₂R), 2.2 (t, 2H,RCH₂COOH), 1.5-18 (bulk methylenes), 1.2-1.4 (bulk methylenes), 0.92 (t,J=7.2 Hz, 9H, methyls). Electrospray (neg. ion) MS [MeOH/H₂O: 95/5](peak, rel. int.) 683 (50), 977.8 (30), 992.1 (M⁻¹, 100), 1008.1(M⁻¹+16, 40), 1024.0 (M⁻¹+32, 10); calc. M⁻¹=992.5 amu. The +16 and +32peaks suggest oxidation of thioethers to sulfoxide groups. (14) has alsobeen successfully synthesized from (13) using (10) in a similar fashionas that described in the synthesis of (11).

[0273] 1-(1,3-dibutylthiobarbiturate)-3-(1-butyl-3-(12-(12-pentylthio-1-dodecylthio)-N-hydroxysuccinimidedodecanoate) thiobarbiturate) trimethineoxonol (15): 22.5 μmol of (14)was reacted with disuccinimidyl carbonate (57 mg, 225 μmol) in 0.5 mLCH₂Cl₂ in the presence of DIEA (39 uL, 225 mmol). After 1.5 hours, TLC(EtOAc/MeOH) (9:1) indicated that 3 new non-polar bands had been formed.The solvent was removed and two non-polar oxonol bands were purfied byflash chromatagraphy, eluting with (EtOAc/MeOH) (95:5). Electrospray(neg. ion) MS [MeOH/H₂O: 95/5] (peak, rel. int.) 1089.3 (M⁻¹, 20),1105.1 (M⁻¹+16, 100), 1121.0 (M⁻¹+32, 60); calc. M⁻¹=1089.5 amu.

Example IV

[0274] Measurement of Membrane Potential with Oxonol Dyes as FRETAcceptors and Fluorescent Lectins as FRET Donors

[0275] FL-WGA was purchased from Sigma Chemical Co. (St. Louis, Mo.).TR-WGA was prepared from WGA and Texas Red (Molecular Probes; Eugene,OR) in a 100 mM bicine buffer at pH 8.5. A 73 μM solution of WGA wasreacted with a 6-fold excess of Texas Red for 1 h at room temperature.The protein conjugate was purified on a G-25 Sephadex column.

[0276] All cells were grown and handled like L-M(TK⁻) except wherenoted. L-M(TK⁻) cells were grown in Dulbecco's Modified Eagle Media(Gibco; Grand Island, N.Y.) with 10% fetal bovine serum (FBS) and 1%penicillin streptomycin (PS) (Gemini; Calabasas, Calif.). B104 cellswere differentiated with 1 μM retinoic acid for 5 days prior to use. Thecells were plated on glass coverslips at least one day before use. Theadherent cells were washed and maintained in 2.5-3.0 mL of HBSS with 1g/L glucose and 20 mM HEPES at pH 7.4. A freshly prepared 75 μM aqueoussolution of the appropriate oxonol was made prior to an experiment froma DMSO stock solution. The cells were stained by mixing 100 μL of theoxonol solution with 750 μL of the bath and then adding the dilutedsolution to the cells. The dye was left for 30-40 minutes at a bathconcentration of 2.3 μM. 1.5 mM β-cyclodextrin in the bath solution wasnecessary for cell loading of DiSBA-C₆-(3). The butyl and ethylderivatives were water-soluble enough to load cells with outβ-cyclodextrin complexation. DiSBA-C₁₀-(3) was loaded in a pH 7.4solution containing 290 mM sucrose and 10 mM HEPES, 364 mOsm, for 10 minat a bath concentration of 10 μM. DiSBA-C₁₀-(3) labeling was quenched byreplacing the bath with HBSS solution. The cells were stained with 15μg/mL of FL-WGA for 15 minutes. The B104 cells required a 125 μg/mL bathconcentration to give satisfactory lectin staining. The excess dyes wereremoved with repeated washes with HBSS. If the excess ethyl or butyloxonol derivatives were left in the bath, slow currents and fluorescencechanges due to redistribution of the dyes into the cell were observedduring depolarizations greater than 1 s. The cardiac myocytes[Henderson, S. A., Spencer, M., Sen, A., Kumar, C., Siddiqui, M. A. Q.,and Chien, K. R. 1989. Structure organization, and expression of the ratcardiac myosin light chain-2 gene. J. Biol. Chem. 264:18142-18146] werea gift of Professor Kenneth Chien, UCSD. The Jurkat lymphocytesuspensions were grown in RPMI media with 5% heat inactivated FBS and 1%PS. 15-20 mL aliquots of the cellular suspension were washed three timesbefore and after dye staining by centrifugation at 100×g for 4 minutesfollowed by additions of fresh HBSS.

[0277] The fluorescently labeled cells were excited with light from a75W xenon lamp passed through 450-490 nm excitation interferencefilters. The light was reflected onto the sample using a 505 nmdichroic. The emitted light was collected with a 63× Zeiss (1.25 or 1.4numerical aperture) lens, passed through a 505 nm long pass filter anddirected to a G-1B 550 nm dichroic (Omega; Brattleboro, Vt.). Thereflected light from this second dichroic was passed through a 515 DF35bandpass filter any made up the FL-WGA signal. The transmitted light waspassed through a 560 or 570 LP filter and comprised the oxonol signal.For experiments using the oxonol as a donor to TR-WGA, the 550 nmdichroic was used for excitation and a 580 nm dichroic was used to splitthe emission. The long wavelength Texas Red fluorescence was passedthrough a 605 nm DF55 bandpass filter. Voltage dependent fluorescencechanges in single cells were measured using a Nikon microscope attachedto a Photoscan II photometer equipped with two R928 PMTs for dualemission recordings. A 7-point Savitsky-Golay smoothing routine wasapplied to all optical data [Savitsky, A. and Golay, M. J. E. 1964.Smoothing and differentiation of data by simplified least squaresprocedure. Anal. Chem. 36:1627-1639], unless otherwise noted. The 1-2KHz single wavelength data was acquired with an Axobasic program thatused the TTL pulse counting routine LEVOKE. Confocal images wereacquired using a home built high speed confocal microscope [Tsien, R. Y.and B. J. Bacskai. 1994. Video-rate confocal microscopy. In Handbook ofBiological Confocal Microscopy. J. B. Pawley, editor. Plenum Press, NewYork]. The cell was voltage-clamped at a holding potential of −70 mV.After a 200 ms delay, the cell was given a 200 ms depolarizing squarevoltage pulse to 50 mV. Pseudocolor images showing the ratio of theFl-WGA to oxonol emissions were collected every 67 ms and clearly showeda change in ratio, localized to the plasma membrane, upon depolarizationof the cell to +50 mV.

[0278] Patch clamp recording were made using an Axopatch 1-D amplifierequipped with a CV-4 headstage from Axon Instruments (Foster City,Calif.). The data were digitized and stored using the PCLAMP software.The pH 7.4 intracellular solution used contained 125 mM potassiumgluconate, 1 mM CaCl₂·2H₂O, 2 mM MgCl₂·6H₂O, 11 mM EGTA, and 10 mMHEPES. For the B104 cells, 4 mM ATP and 0.5 mM GTP were added.

[0279] The quantum yield of DiSBA-C₆-(3) was determined relative torhodamine B in ethanol (_(F)=0.97) [Weber, G. and Teale, F. W. K. 1957.Determination of the absolute quantum yield of fluorescent solutions.Faraday Soc. Trans. 53:646-655]. R_(o) was calculated following standardprocedures [Wu, P. and Brand, L. 1994. Resonance energy transfer:methods and applications. Anal. Biochem. 218:1-13]. The spectra ofFL-WGA in HBSS and DiSBA-C₆-(3) in octanol were used to determine theoverlap integral. Values of 1.4 and 0.67 were used for the index ofrefraction and orientation factor respectively.

[0280] Symmetrical bis(thiobarbiturate)oxonols were chosen as likelycandidates for rapidly translocating fluorescent ions based on the abovedesign criteria. The strong absorbance maximum (⁻200,000 M⁻¹cm⁻¹) at 540nm and good quantum yield (0.40) in membranes makes them desirable foruse as a fluorescence donors or acceptors in cells. The fluorescenceexcitation and emission spectra of DiSBA-C₆-(3) is shown in FIG. 2 alongwith those for FL-WGA and TR-WGA. The excitation spectra are the shorterof each pair. Octanol was selected as the oxonol solvent in order tomimic the membrane environment.

[0281] The translocation rates were studied in L-M(TK⁻) cells usingwhole-cell voltage clamp recording. The L-M(TK⁻) cells were chosenbecause they have very low background currents and are easy to patchclamp. These cells have a resting potential of −5 mV and no evidentvoltage activated currents.

[0282] Displacement currents from DiSBA-C₆-(3) at 20° C. are displayedin FIG. 3. 12.5 ms long voltage steps at 15 mV increments were appliedto the cell, from a holding potential of −70 mV. The larger, fastertransients due to simple membrane capacitance transient could beminimized using the capacitance and series resistance compensationcapabilities of the Axopatch amplifier, allowing the displacementcurrents to be clearly observed. The currents are due to redistributionof the membrane-bound oxonol in response to 8 depolarizations. The timeconstant for the displacement current is 2 ms for 120 mV depolarization.Equal amounts of charge move at the onset and conclusion of the voltagestep, but in opposite directions, consistent with redistribution ofstored ions from one energy minimum to the other across the plasmamembrane. Furthermore, the induced capacitance dq/dV from the oxonolmovement is calculated to be ⁻5 pF for 100 mV depolarization. This valuecorresponds to roughly one third the membrane capacitance without thedye. Interestingly, sodium channel gating charges are also responsiblefor about 33% of the total capacitance of squid axons for smalldepolarizations [Hodgkin, A. 1975. The optimum density of sodiumchannels in an unmyelinated nerve. Philos. Trans. R. Soc. Lond. [Biol]270:297-300]. Negligible currents were observed in the absence of theoxonol. DiSBA-C₁₀-(3) gave displacement currents of approximately thesame speed, whereas analogues with R=butyl and ethyl gave much slowercurrents. The butyl compound had a time constant of ⁻18 ms and thecurrents from the ethyl compound were very small, slow, and difficult toobserve.

[0283]FIGS. 4 and 5 show the voltage dependence and time constants forcharge translocation in a cell loaded with about 4 times as much oxonolas in the experiment of FIG. 3. In FIG. 4, the circles are the data fromthe on response and the squares from the tail currents. The raw datawere fit to a single exponential and the charge moved, the area, wascalculated as the product of the current amplitude and the timeconstant. The experimental data are in reasonable accord with existingmodels of hydrophobic ion transport between two energy minima near theaqueous interfaces of the lipid bilayer [Ketterer, B., Neumcke, B., andLäuger, P. 1971. Transport mechanism of hydrophobic ions through lipidbilayer membranes. J. Membrane Biol. 5:225-245; Andersen, O. S. andFuchs, M. 1975. Potential energy barriers to ion transport within lipidbilayer. Biophys. J. 15:795-830; Benz, R., Läuger, P., and Janko, K.1976. Transport kinetics of hydrophobic ions in lipid bilayer membranes.Biochim. Biophys. Acta 455:701-720]. These models predict that theequilibrium charge displacement q(V) and the translocation time constant(V) should depend on the externally applied membrane potential V in thefollowing manner: $\begin{matrix}{{\Delta \quad {q(V)}} = {\Delta \quad q_{\max}{\tanh \left\lbrack \frac{q\quad {\beta \left( {V - V_{h}} \right)}}{2\quad {kT}} \right\rbrack}}} & (1) \\{{\tau (V)} = {\tau_{\max}{{sech}\left\lbrack \frac{q\quad {\beta \left( {V - V_{h}} \right)}}{2{kT}} \right\rbrack}}} & (2)\end{matrix}$

[0284] V_(h), the membrane potential at which there are equal numbers ofions in each potential energy well, could differ from zero because ofmembrane asymmetry. β is the fraction of the externally appliedpotential effectively felt by the translocating ion; q is the charge oneach ion, k and T are Boltzmann's constant and absolute temperature.q_(max) and τ_(max) are respectively the total charge in each energywell and the time constant for translocation, both at V=V_(h). Thesmooth curve in FIG. 4 is the fit to Eq. 1 with q_(max)=4770±140 fC,β=0.42±0.02, and V_(h)=−3.8±1.5 mV. Likewise the smooth curve in FIG. 5is the fit to Eq. 2 with τ_(max)=2.9 ms at V_(h)=−5 mV and β=0.42.

[0285] These results demonstrate that the oxonol senses a significantpart of the electric field across the membrane, that it translocates in⁻3 ms or less, and that the greatest sensitivity and linearity oftranslocation as a function of membrane potential is in thephysiologically relevant range.

[0286] To transduce charge displacements into optical signals, theoxonol fluorescences at the intracellular and extracellular membranebinding sites is made different. Fluorescence asymmetry is created withthe introduction of fluorescently labeled lectins bound to theextracellular membrane surface. Excitation of FL-WGA leads to energytransfer to oxonols located in the extracellular membrane binding siteas shown in FIG. 1. The extinction coefficient and the fluorescencequantum yield of FL-WGA were measured to be 222,000 M⁻¹cm⁻¹ (⁻3fluorescein/protein) and 0.23, respectively. In Jurkat cell suspensionslabeled with FL-WGA, up to 30% of the lectin fluorescence intensity wasquenched upon titration of DiSBA-C₄-(3). In the best case where all ofthe quenching is due to energy transfer, the average distance from thelectin to the membrane-bound oxonol is still greater than 50 Å, thecalculated Forster distance R_(o) for the FL-WGA/oxonol pair. Thespectral overlap between the FL-WGA emission and DiSBA-C₆-(3) excitationis given in FIG. 2. Because FRET falls off with the inverse sixth powerof the distance separating the two fluorophores, energy transfer tooxonols in the intracellular membrane site, an additional 40 Å away, isprobably negligible.

[0287] Upon depolarization, the oxonol molecules redistribute such thatmore are bound to the intracellular site and less to the extracellularone. This change manifests itself with a decrease in the energytransfer, resulting in an increase in the fluorescence of the FL-WGA anda concomitant decrease in the oxonol emission. The fluorescence signalsin a voltage clamped L-M(TK⁻) cell labeled with (the DiSBA-C₄-(3)/FL-WGApair) and depolarized with four increasing voltage steps are shown inFIG. 6. The data are the average of 29 sweeps. The FL-WGA emissionincreases 7-8%, the oxonol fluorescence decreases 10% and theFL-WGA/oxonol emission ratio changes 19% for a 120 mV depolarization.The simultaneous changes in donor and acceptor emissions is consistentwith the FRET mechanism outlined in FIG. 1. The decrease in oxonolemission with depolarization is opposite to what is observed for theslow voltage-sensitive uptake of oxonols in cells [Rink et al. 1980,supra]. The fluorescence changes have time constants of ⁻18 ms at 20°C., in agreement with the DiSBA-C₄-(3) displacement currents. No largefluorescence changes are observed in the absence of FL-WGA. Thetranslocation rate of DiSBA-C₄-(3) speeds up with increasingtemperature. The time constant falls to 7-8 ms at 29° C., correspondingto an activation energy of ⁻17 kcal/mol. However, raising thetemperature also increases internalization of the lectin and eventuallydecreases the fluorescence change. The oxonols with R=ethyl and butylalso reach internal cellular membranes, though active membraneinternalization is probably not necessary. Additional dilution of thevoltage-dependent FRET signals arises from spectral overlap of thefluorescein and oxonol, such that some of the light in the fluoresceinemission channel comes from the oxonol and vice versa.

[0288] Increasing the length of the alkyl chains on the oxonol improvesthe response times significantly. The DiSBA-C₆-(3)/FL-WGA pair, has atime constant of ⁻3 ms at 20° C., while the DiSBA-C₁₀-(3)/FL-WGA pair,responds with a time constant of 2 ms, as shown in FIG. 7. The solidcurve is a fit to a single exponential with a 2 ms time constant. Thedata is the average of 3 L-M(TK⁻) cells, at 20° C., acquired at 2 kHz.The response in the figure is slightly slower than the true valuebecause of smoothing. The fluorescence time constants are in agreementwith those from the displacement currents, for example in FIG. 3. Thebeneficial effect of adding hydrophobicity to the oxonol in the form oflonger alkyl chains reaches a plateau. There is a large 6-fold increasein translocation rate substituting hexyl for butyl on the oxonol core.However, addition of twice as many methylene groups in going from thehexyl to the decyl compound results in less than a 2-fold increase.These faster translocating oxonols are essentially insoluble in waterand require modified procedures to load into cells. DiSBA-C₆-(3) iseasily loaded in normal medium supplemented with 1.5 mM β-cyclodextrinto complex the alkyl chains. Nonfluorescent DiSBA-C₆-(3) aggregates inHanks Balanced Salt Solution (HBSS) become fluorescent upon addition ofβ-cyclodextrin. DiSBA-C₁₀-(3) requires loading in a medium of low ionicstrength with osmolarity maintained with sucrose. Labeling is confinedalmost exclusively to the plasma membrane, presumably because thehydrophobicity is now great enough to prevent desorption from the firstmembrane the dye encounters.

Example V

[0289] Measurement of Membrane Potential with Oxonol Dyes as FRETAcceptors and Fluorescent Lipid FRET Donors

[0290] A.Trimethine oxonols

[0291] A 6-chloro-7-hydroxycoumarin conjugated todimyristoylphosphatidylethanolamine (DMPE) via a glycine linker, Cou-PE,has been prepared and found to function as a excellent voltage-sensitiveFRET donor to bis-(1,3-dialkyl-2-thiobarbiturate)-trimethineoxonols.This new FRET pair has given an 80% ratio change for a 100 mVdepolarization in an astrocytoma cell, which is the largestvoltage-sensitive optical signal observed in a cell, FIG. 17. Thevoltage sensitivity of this FRET pair is consistently 2-3 times betterthan Fl-WGA/trimethineoxonol in a variety of cell types. In L-cells,ratio values between 25-50% are found, with equal percent changes foundin both channels, FIG. 18. In neonatal cardiomyocytes, 5-30%fluorescence ratio changes were observed for spontaneously generatedaction potentials. The largest signals are almost 4 times larger thanthose possible with Fl-WGA/trimethineoxonol. An example of such a largechange from a single cluster of heart cells is given in FIG. 19. Thebenefits of a ratio output are evident in the figure. The individualwavelengths, at the top of the figure, show a decreasing baseline thatis due to fluorophore bleaching. The ratio data shown on the bottom ofthe figure compensates for the loss of intensity in both channels andresults in a flat baseline. Furthermore, motion artificially causesbroadening of the individual wavelength responses. The ratio datareduces these artifacts and results in a sharper signal that moreclosely represents the actual voltage changes. The greater sensitivityfor this new FRET pair is most likely due to a combination of factors.Moving the donor closer to the membrane surface and decreasing theForster transfer distance, R_(o), may result in increased FRETdiscrimination between the mobile ions on the same and opposite sides ofthe membrane. Also, the increased spectral separation facilitatescollection of the donor and acceptor emission and reduces signal lossdue to crosstalk.

[0292] B.Pentamethine oxonols

[0293] Bis-(1,3-dialkyl-2-thiobarbiturate)-pentamethineoxonols have beenprepared by condensing 1,3 substituted thiobarbituric acids withglutacondialdehyde dianil monohydrochloride, also known asN-[5-(phenylamino)-2,4-pentadienylidene]benzenamine monohydrochloride.The pentamethine oxonols absorb at 638 nm (e=225,000 M⁻¹ cm⁻¹) andfluoresce maximally at 663 nm in ethanol. The absorbance and emissionare shifted 100 nm to longer wavelengths, compared to the trimethineoxonols. This shift is consistent with other polymethine dyes, such ascyanines, where addition of 2 additional methine units results in 100 nmwavelengths shifts to the red.

[0294] The pentamethines can be loaded into cells in culture in the samemanner as the trimethine oxonol. The butyl compound, DiSBAC₄(5), can beloaded in Hanks' Balanced Salt Solution , while the hexyl compound,DiSBAC₆(5), requires addition of beta-cyclodextrin to mask the hexylside chains and solubilize the hydrophobic oxonol.

[0295] Voltage-sensitive FRET from various plasma membrane-boundfluorescent donors to the pentamethine oxonols have been demonstrated insingle cells. Fl-WGA has been shown to undergo FRET with thepentamethine and give ratio changes comparable to those observed for thetrimethine oxonol. In astrocytoma cells, ratio changes of 15-30% wererecorded for a 100 mV depolarization step from −70 mV. Apparently, thedecrease in FRET due to the reduced overlap integral, J, on moving theoxonol absorbance 100 nm longer is compensated by increased selectivityof FRET to the extracellular face of the membrane relative to theintracellular one and/or decreased spectral overlap.

[0296] Fluorescent phosphatidylethanolamine conjugates have also beenfound to function as FRET donors to the pentamethine oxonol. Thestructures of PE conjugates tested are shown in FIG. 20.NBD-PE/pentamethineoxonol pair has given 1-10% ratio changes per 100 mV.Cou-PE/pentamethineoxonol has given 15-30% ratio changes in voltageclamped astrocytomas for 100 mV depolarization. This pair is remarkablebecause the Cou-PE emission and DiSBAC₆(5) absorbance maxima areseparated by 213 nm and there is hardly any visible overlap, FIG. 21.The large extinction at long wavelengths of the pentamethine enable FRETbetween the coumarin and the pentamethine oxonol. The R_(o) for thispair has been calculated to be 37 Å, using a quantum yield value of 1.0for the Cou-PE.

[0297] The membrane translocation rates for the pentamethines are 5-8times faster than the trimethine analogues. DiSBAC₄(5) displacementcurrents in voltage-clamped astrocytoma cells showed that butylpentamethine oxonol jumps across the plasma membrane with a timeconstant of ⁻2 ms in response to voltage steps of +20-120 mV. Thetrimethine analogue translocates ⁻18 ms under identical conditions. Thedisplacement currents of DiSBAC₆(5) decay very rapidly and are difficultto separate from the cell capacitance. As a result of the largevoltage-dependent signal from the Cou-PE/ DiSBAC₆(5) pair, it waspossible to optically measure the speed of voltage response ofDiSBAC₆(5). The time constant for DiSBAC₆(5) translocation was measuredoptically at 0.383±0.032 ms in response to a 100 mV depolarization step,using FRET from asymmetrically labeled Cou-PE. The ratio data and theexponential response are shown in FIG. 22. The enhanced translocationrates result from the greater charge delocalization and slightly morehydrophobicity of the pentamethine oxonols. The rapid voltage responseof DiSBAC₆(5) is the fastest known for a permeant, highly fluorescentmolecule. The submillisecond response is fast enough to accuratelyregister action potentials of single neurons.

Example VI

[0298] Measurement of Membrane Potential with Oxonol Dyes as FRET Donors

[0299] The direction of energy transfer can be reversed using TR-WGAinstead of FL-WGA. DiSBA-C₆-(3) functions as a FRET donor to TR-WGA inL-M(TK⁻) cells with the same response time as FL-WGA/DiSBA-C₆-(3). Thespectral overlap of this FRET pair is shown in FIG. 2. The signalchange, however, is only one half that for FL-WGA/DiSBA-C₆-(3).

[0300] DiSBAC₆(3) has been successfully used as a FRET donor to Cy5labeled PE, in B104 neuroblastoma cells. The ratio changes of 5-15%/100mV are the largest observed with the mobile ion being the donor.

Example VII

[0301] Measurement of Membrane Potentials Different Cell Types

[0302] The FL-WGA/DiSBA-C₆-(3) system was tested in a variety of celllines. In neonatal cardiac myocytes, the two fluorophores could beloaded without affecting the spontaneous beating. Therefore, the addedcapacitance from the oxonol displacement current did not preventgeneration of action potentials. The periodic 90 mV action potentials[Conforti, L., Tohse, N., and Sperelakis, N. 1991. Influence ofsympathetic innervation on the membrane electrical properties ofneonatal rat cardiomyocytes in culture. J. Devel. Physiol. 15:237-246]could be observed in a single sweep, FIG. 8C. The ratio change withoutsubtraction of any background fluorescence was 4-8%. Motion artifactswere observed in the single wavelength data. In FIG. 8A and B, largeslow changes in the detected light were observed in both channels.Satisfyingly, these effects were essentially eliminated in the ratiodata. The data were acquired at 100 Hz and 10 μM of isoproterenol wasadded to the bath solution. The voltage dependent fluorescence changesare faster than the mechanically based artifacts, as expected [Hill, B.C. and Courtney, K. R. 1982. Voltage-sensitive dyes discerningcontraction and electrical signals in myocardium. Biophys. J.40:255-257]. Some cells, loaded with oxonol at 2.3 μM, did stop beatingafter about 7 seconds of continuous exposure to the xenon arcillumination. At 0.6 μM loading, the phototoxicity and unfortunately thesignal were reduced. In differentiated B104 neuroblastoma cells an 8%ratio increase was recorded, without any background subtraction, for a120 mV depolarization. The inward sodium currents did not deterioratefrom phototoxic effects during experiments with excitation exposurestotaling 10-20 s. FL-WGA/DiSBA-C₆-(3) labeled 1321N astrocytoma cellsshowed oxonol and FL-WGA fluorescence almost exclusively on the plasmamembrane. Ratio changes of 22-34% for 100 mV were observed in photometryexperiments such as FIG. 9. After a 50 ms delay, the membrane potentialwas depolarized 100 mV from −70 mV for 100 ms. The traces are theaverage of 4 sweeps acquired at 300 Hz, with no smoothing. The timeconstant for the fluorescence changes is less than 3.3 ms consistentwith the displacement currents, such as those in FIG. 3. A smallbackground signal was subtracted from the starting signal, <5% for theoxonol channel and <15% for the fluorescein channel. The fluorescenceintensities in the fluorescein and oxonol channels increased ⁻17% anddecreased ⁻16% respectively for 100 mV depolarization. In these cells,unlike the L-M(TK⁻), the crosstalk between emission channels wasdecreased and larger changes occurred in the fluorescein signal. Thesesignal changes are the largest millisecond membrane potential dependentratio changes observed in single cells. Previous investigations haveshown that 4-ANEPPS gives a 9%/100 mV excitation ratio change [Montana,V., Farkas, D. L., and Loew, L. M. 1989. Dual-wavelength ratiometricfluorescence measurements of membrane potential. Biochemistry28:4536-4539]. In addition, FL-WGA/DiSBA-C₆-(3) fluorescence changes ineach emission channel are comparable to the largest reported changes,for example, the 21%/100 mV change in a neuroblastoma using RH-421[Grinvald, A., Fine, A., Farber, I. C., and Hildesheim, R. 1983.Fluorescence monitoring of electrical responses from small neurons andtheir processes. Biophys. J. 42:195-198]. The large signals fromFL-WGA/DiSBA-C₆-(3) made it possible to record ratio images of membranepotential changes in voltage clamped L-M(TK⁻) and astrocytoma cellsusing a high speed confocal microscope.

[0303] The astrocytoma cells gave a 10-20% ratio increase that waslocalized to the plasma membrane for a 120 mV depolarization.

Example VIII

[0304] Synthesis of Fluorescent Tetraaryl Borates

[0305] With reference to FIG. 10, in a 25 mL flame dried two neck flask0.788 g (580 μL, 2.2 mmol) of compound I was dissolved in 6 mL of dryhexane under argon. After cooling the flask to −70 C., 1.46 mL of a 1.5M n-butyllithium solution (2.2 mmol) was added via syringe. In aseparate flask 0.60 dry borane II was dissolved in a deoxygenatedmixture of 6 mL hexane and 1.5 mL of freshly distilled THF. The boranesolution was then added via syringe to the lithium reagent. A solidimmediately precipitated. After 30 min the cold bath was removed and thereaction was allowed to slowly heat up. Three hours later the solventwas decanted off and the solid rinsed with more hexane. The solid wasdissolved in acetonitrile and water and then poured into a separatoryfunnel. The aqueous layer was again washed with hexane and thenextracted with ethyl acetate. Half of the extract was concentratedyielding 124.9 mg ( 170.5 μmol) of the desired product. This product wasthen mixed with 97 mg of tetrabutylammonium fluoride in acetonitrile for15 min at room temperature. After workup 129.1 mg of compound III as thetetrabutylammonium salt was recovered (93 %). ¹HNMR (d₆ acetone) 7.61(br d, 2H, CF₃-phenyl group), 7.43 (cm, ⁻3H, CF₃-phenyl group),6.90-7.26 (cm, ⁻7H, CF₃-phenyl group), 3.43 (cm, 8H, NCH ₂CH₂ CH₂ CH₃),1.81 (cm, ⁻8H, NCH₂ CH₂ CH₂ CH₃), 1.42 (cm, ⁻8H, NCH₂CH₂ CH ₂ CH₃), 0.93(t, J=7.1 Hz, NCH₂CH₂ CH₂ CH ₃).

[0306] With reference to FIG. 10, for synthesis of compound IV, in a 5mL round bottom flask 14 mg (17.2 umol) of compound III, 7 mg (25.8umol) of bromomethylbimane, 27.3 mg (172 umol) of potassium carbonate,and 5 mg (18.9 umol) of 18-crown-6 were mixed in 0.6 mL of dryacetonitrile. The mixture was heated at 70 C. 1.5 h. After cooling, thereaction mixture was dissolved in ethyl acetate and washed 3× withwater. The organic residue was purified by flash chromatography elutingwith toluene/acetone (2:1). The major band was collected yielding 12.1mg (70%) of pure product IV tetrabutyl ammonium salt. ¹HNMR (d₆ acetone)7.58 (br d, 2H, CF₃-phenyl group), 7.4-7.5 (cm, 2H, CF₃-phenyl group),7.0-7.3 (cm, ⁻10H, CF₃-phenyl group), 5.29 (d, J=1.6 Hz, 2H, CH₂), 3.46(cm, 8H, NCH ₂CH₂ CH₂ CH₃), 2.56 (d, J=0.7 Hz, 3H, bimane methyl), 1.84(cm, ⁻8H, NCH₂ CH₂ CH₂ CH₃), 1.79 (d, J=0.8 Hz, 3H, bimane methyl), 1.76(s, 3H, bimane methyl), 1.44 (cm, ⁻8H, NCH₂CH₂ CH ₂ CH₃), 0.98 (t, J=7.2Hz, NCH₂CH₂ CH₂ CH ₃); _(f)=0.73 in dioxane based on quinine sulfate in0.1 N H₂SO₄ _(f)=0.55.

Example IX

[0307] Synthesis of Asymmetric Oxonols with a Linker Group

[0308] (A)This example illustrates the synthesis of asymmetric oxonolscontaining a built-in linker group. With reference to FIG. 11, forsynthesis of compound V, 4.35 g (21.7 mmol) of 1,12-diaminododecane wasdissolved in 40 mL of dry CH₂Cl₂. Via syringe, 2.62 mL (2.17 mmol) ofbutyl isothiocyanate was added to the reaction flask. A white solid hadprecipitated after 15 minutes. One hour after the addition, the reactionmixture was filtered. The filtrate was then evaporated leaving a whitesolid. The solid was redissolved in 45 mL of dry CH₂Cl₂ and mixed with2.44 mL of N,N-diisopropylethylamine (DIEA) and 3.9 g (17.9 mmol) ofdi-tert-butyl dicarbonate. After reacting for 1 hour, the mixture waspoured into a separatory funnel and washed with 5% sodium bicarbonate. Asolid came out of solution and was filtered away (<100 mg). The organicsolution was then washed with water and a saturated brine solution. Theorganic layer was then dried with MgSO₄ and filtered. The filtrate wasevaporated leaving a white solid, which was recrystallized in isopropylether yielding 4.30 g (10.3 mmol) of pure compound V (48% overall).¹HNMR (CDCl₃) 5.73 (br s, 2H, thioamide), 4.50 (br s, 1H, carbamate),3.40 (br s, 4H, NCH ₂), 3.10 (q, J=7.2 Hz, ⁻3 H, CH ₂ next tocarbamate), 1.44 (s, 9H, t-butyl), 1.2-1.7 (cm, bulk CH ₂ s), 0.94 (t,J=7.2 Hz, n-butyl methyl).

[0309] For preparing compound VI, 441 mg (19.2 mmol) of sodium wasdissolved in 5 mL of dry ethanol under argon. When almost all of thesodium was dissolved, 2.92 mL (3.1 g, 19.2 mmol) of diethyl malonate wasadded to the ethoxide solution. Some solid precipitated out of solution.4.0 g (9.6 mmol) of compound V was added and the mixture was refluxedunder argon at 100 C. for 70 hours. After cooling, the reaction mixturewas filtered and washed with ethanol. Water was added to the filtrateand a white solid precipitated out of solution. The solid (779 mg,mostly of unreacted starting material) was filtered away. The filtratewas then acidified to pH ⁻2 and then extracted into ethyl acetate. Theorganic layer was then dried with MgSO₄ and filtered. After removing thesolvent, 1.6 g (3.3 mmol) of a yellow oil was recovered (34%). ¹HNMR(CDCl₃) 4.22 (cm, 4H, NCH ₂ next to barbiturate). 3.63 (s, 2H, ring CH₂), 2.99 (cm, 2H, CH ₂ next to carbamate), 1.53 (cm, 4H, NCH₂CH ₂), 1.34(s, 9H, t-butyl), 1.1-1.3 (cm, bulk CH ₂ s), 0.85 (t, J=7.4 Hz, n-butylmethyl).

[0310] To prepare compound VII, 1 mL of trifluoroacetic acid (TFA) wasadded with stirring to 200 mg (0.41 mmol) of compound VII dissolved in 3mL of CH₂Cl₂. After 1.25 hours, all the solvent was removed underreduced pressure. One equivalent each ofN-[5-(phenylamino)-2,4-pentadienylidene]aniline monohydrochloride and1,3-di-n-butylthiobarbiturate was added and all three componentsdissolved in 1 mL pyridine and left overnight. The product was purifiedfrom the other pentamethine oxonols by flash chromatography. Thenonpolar products were eluted with CHCl₃/CH₃OH (9:1). The pure productcontaining the linker eluted with CHCl₃/CH₃OH (1:1). The product wasbound very tightly to the silica gel and only 10 mg of product wasrecovered. ¹HNMR (CDCl₃/CD₃OD) 7.5-7.8 (cm, 4H, vinyl methines), 7.35(t, J=⁻14 Hz, 1H, central methine), 4.34 (br t, ⁻10 H, NCH ₂ next tobarbiturate), 2.72 (cm, ⁻3H, CH ₂ next to amine), 1.4-1.7 (br cm, ⁻12H,NCH₂CH ₂), 1.0-1.4 (cm, ⁻40H, bulk CH ₂ s), 0.81 (t, J=7.3 Hz, 9H,n-butyl methyl).

[0311] (B)This particular example is with reference to FIGS. 15 and 16.

[0312] N-butyl-N-5-pentanol thiourea (6):5-amino-1-pentanol (1.416 mL,13 mmol) was dissolved in 7 mL of CH₂Cl₂. Under argon,butylisothiocynate (1.570 mL, 13 mmol) was added via syringe. After 2.5h, the solvent was removed under vacuum leaving an oil. Under highvacuum, the oil was freeze dried 2× with liquid nitrogen and left underreduced pressure over night. The next morning 2.615 g of pure solidproduct was collected (12 mmol, 92%). ¹H NMR ( CDCl₃): d 5.90 (br s, 2H,NH), 3.66 (t, J=6.2 Hz, 2H, RCH₂OH), 3.42 (br m, 4H, NHCH₂R), 1.80 ( brs, 1H, OH), 1.3-1.7 (unres. cm's, 10H, bulk methylenes), 0.94 (t, J=7.2Hz, 3H, methyl). ¹³C NMR (CDCl₃): d 181.5 (thiocarbonyl), 62.2 (RCH₂OH),44.2 (NHCH₂R), 44.1 (NHCH₂R), 31.9 (CH₂), 31.0 (CH₂), 28.6 (CH₂), 23.0(CH₂), 19.9 (CH₂), 13.6 (CH₃).

[0313] 1-butyl,3-(5-pentanol) thiobarbiturate (7):In dry EtOH, 345 mg(15 mmol) of Na was dissolved. After almost all of the Na had dissolved,diethylmalonate (2.278 mL, 15 mmol) was added under argon. The mixturewas then heated to 60° C. to dissolve the precipitated sodium malonate.The heat was then removed and N-butyl-N-5-pentanol thiourea (6) (1.310g, 6 mmol) was added. The reaction mixture was refluxed a 100° C. for3.5 days. After cooling, the reaction mixture was filtered and washedwith EtOH. An approximately equal volume of H₂O was added to thefiltrate and acidified to pH 1-2 with conc. HCL. The aqueous solutionwas extracted 3× with 1:1 EtOAc/hexanes. The combined extracts weredried with MgSO₄, filtered, and concentrated leaving an oil. TLCEtOAc/MeOH (4:1) showed that in addition to the major barbiturateproduct there were two nonpolar impurities. Flash silica gelchromatography afforded some purification (4×17 cm), eluting withEtOAc/MeOH (4:1). The material was still an oil and a second column wasdone eluting with CHCl₃/MeOH/AA (90:8:2). Despite being an oil, 0.726 g(2.54 mmol, 42%) of pure product was recovered. ¹H NMR (CDCl₃): d 4.31(cm, 4H, NCH₂R), 3.71 (br s, 2H, ring methylene), 3.63 (t, J=6.3 Hz, 2H,RCH₂OH), 2.75 ( br s, 1H, OH), 1.5-1.8 (cm, 6H, bulk methylenes),1.2-1.5 (cm, 4H, bulk methylenes), 0.94 (t, J=7.2 Hz, 3H, methyl).

[0314] 1-butyl,3-(5-bromopentane) thiobarbiturate (8): (7) (98 mg, 343umol) was dissolve in 600 uL dry CH₂Cl₂ and mixed with carbontetrabromide (142 mg, 429 umol). The solution was cooled on ice andtriphenylphosphine (135 mg, 515 umol) was added. The solution bubbledand turned yellow immediately. After 30 min. the solvent was removed andhexane was added to the solid residue. The mixture was allowed to stirovernight. TLC showed only 1 barbiturate in hexane solution along withtriphenylphospine oxide. The impurity was removed by flash silica gelchromatography (2.5×22 cm) packed in EtOAc/MeOH (98:2). The nonpolarimpurity was eluted off the column using the packing solvent followed byEtOAc/MeOH (90:10). The desired product was eluted with CHCl₃/MeOH/AA(93:5:2), yeilding 40 mg (115 umol, 34%). ¹H NMR (CDCl₃): d 4.33 (cm,4H, NCH₂R), 3.72 (s, 2H, ring methylene), 3.42 (t, J=6.7 Hz, 2H,RCH₂Br), 1.91 (cm, 2H, methylene), 1.66 (cm, 4H, methylenes), 1.52 (cm,2H, methylene) 0.95 (t, J=7.2 Hz, 3H, methyl).

[0315] 1,3-di-butyl-5-(3-phenylamino propendienyl) thiobarbiturate(10):Malonaldehyde bis(phenylimine) (500 mg, 1.69 mmol) was dissolved in20 mL of dry DMF. Separately, 1,3 di-butyl thiobarbiturate (430 mg, 1.76mmol) was dissolved in 5 mL dry pyridine and placed in a 10 mL droppingfunnel. The thiobarbiturate solution was slowly added over 5 min and thereaction was left stirring for 5 h. About 20 mL of water was added tothe mixture and a yellow solid precipitated out of solution. The solidwas filtered and dried yielding 575 mg (1.5 mmol, 89%). Minorimpurities, including oxonol, were removed by flash silica gelchromatography (3×15 cm) eluting with EtOAc/hexanes (1:1). Some materialprecipitated on the column. Neverth.elessr after drying, 390 mg of pureproduct was recovered (1 mmol, 59%). ¹H NMR ( CDCl₃/MeOH): d 8.10 (d,J=3.0 Hz, 1H), 8.04 (s, 1H), 7.3-7.5 (cm, 3H), 7.1-7.25 (cm, 3H), 4.40(cm, 4H, NCH₂R), 1.65 (cm, 4H, NCH₂CH₂R), 1.36 (cm, 4H, NCH₂CH₂CH₂CH₃),0.90 (t, J=7.3 Hz, 6H, methyls).

[0316] 1-(1,3-dibutylthiobarbiturate)-3-(1-butyl,3-(5-hydroxypentyl)thiobarbiturate)trimethineoxonol triethylammonium salt (11):Compound (7) (85 mg, 297umol) was mixed with (10) (114 mg, 297 umol) in 1.2 mL dry pyridine andleft stirring for 17 h. TLC EtOAc/MeOH showed that there was 3 majoroxonol products. Conc. HCL was added to 80% of the reaction mixturewhich caused a solid to precipitate out of solution. The solid wasfiltered and washed with water. After drying 220 mg of red solid wasrecovered. 96 mg of this solid was mixed with 1-2 mL EtOAc and filtered.The remaining solid was dissolved in CHCl₃ /MeOH (95:5) and loaded on toa 19×2.5 cm silica gel column. Eluting with the same solvent, the mostnonpolar oxonol was eluted off the column. The solvent was then changedto CHCl₃/MeOH/Et₃N (90:8:2) to elute the middle band which was shown byNMR to be the desired product. After concentrating and drying, 18.5 mg(28 umol, 27%) of the pure product was recovered. ¹H NMR (CDCl₃): d 8.60(t, J=13.9 Hz, 1H, central methine), 8.14 (dd, J₁=13.8 Hz, J₂=2,2 Hz,2H, methines), 4.45 (cm, 8H, NCH₂R), 3.66 (t, J=6.3 Hz, 2H, RCH₂OH),3.20 (q, J=7.3 Hz, 6H, triethylammonium), 1.5-1.8 (cm, 8H, NCH₂CH₂R),1.2-1.5 (cm, 10H, bulk methylenes), 1.35 (t, J=7.3 Hz, 9H,triethylammonium), 0.95 (t, J=7.2 Hz, 9H, terminal methyl). MS.

[0317] 1-(1,3-dibutylthiobarbiturate)-3-(1-butyl-3-(5-tosyloxypentyl)thiobarbiturate)trimethineoxonol (12):In 4 mL pyridine, (11) (86.1 mg, 131 umol) wasmixed with tosyl chloride (333 mg, 1.75 mmol). The reaction mixture wasstirred for 3.5 h before the solvent was removed under vaccuum. Theresidue was dissolved in EtOAc and washed with 1 M HCL, followed by sat.brine 2×. TLC EtOAc/MeOH (9:1) showed that most of the starting materialwas converted to a more non polar product. However, a polar oxonolimpurity was evident and the material had to be further purified byflash chromatography. The column was packed in CHC/₃/MeOH (97:3). It wasnecessary to increase the polarity to CHCl₃/MeOH (92:8) in order toelute off the product. The fractions containg the desired product werecombined and dried, yielding 74.8 mg (102 umol, 78%) of product. ¹H NMR(CDCl₃): d 8.50 (t, 1H, central methine), 7.92 (dd, J₁=14 Hz, J₂=2 Hz,2H, methines), 7.81 (d, 2H, tosyl), 7.35 (d, J=8.2 Hz, 2H, tosyl), 4.37(cm, 8H, NCH₂R), 4.09 (br t, 2H, RCH₂OTs), 2.45 (s, 3H, tosyl methyl),1.2-1.9 (unres. cm, bulk methylenes), 0.91 (t, J=7.2 Hz, 9H, terminalmethyl).

Example IX

[0318] Synthesis of Fluorescent Lanthanide Chelates with a SingleNegative Charge.

[0319] Terbium(III) Bis-(N,N′-bis(salicylidene)ethylenediamine)piperidinium salt, Hpip⁺ Tb(SALEN)₂⁻:N,N′-bis(salicylidene)ethylenediamine (SALEN) (0.719 g, 2.68 mmol) wasdissolved in 40 mL MeOH at 60° C. Terbium chloride hexahydrate (0.5g,1.34 mmol) dissolved in 1 mL water was added to the solution. Piperidine(536 μL, 5.42 mmol) was added and a yellow precipitate immediatelyformed. After 1 h, the heat was removed and the reaction mixture wasleft stirring overnight. The solid was filtered and dried yielding 709mg (0.91 mmol, 68%) of the desired complex. Electrospray (neg. ion) MS[MeOH/H₂O: 95/5] (peak, rel. int.) 691.2 (M⁻¹, 100) calc. M⁻¹=691.5 amu.

[0320] Europium(III) Bis-(N,N′-bis(salicylidene)ethylenediamine)piperidinium, Hpip⁺ EU(SALEN)₂ ⁻:N,N′-bis(salicylidene)ethylenediamine(SALEN) (0.360 g, 1.34 mmol) was dissolved in 40 mL MeOH and piperidine(298 μL, 3.02 mmol) at 60° C. Europium chloride hexahydrate (0.246 g,0.67 mmol) dissolved in 0.5 mL water was added to the solution and ayellow precipitate immediately came out of solution. After 1 h, the heatwas removed and the reaction mixture was left for 2 hours. The solid wasfiltered and dried yielding 341 mg (0.44 mmol, 66%) of the desiredcomplex.

[0321] The foregoing invention has been described in some detail by wayof illustration and example, for purposes of clarity and understanding.It will be obvious to one of skill in the art that changes andmodifications may be practiced within the scope of the appended claims.Therefore, it is to be understood that the above description is intendedto be illustrative and not restrictive. The scope of the inventionshould, therefore, be determined not with reference to the abovedescription, but should instead be determined with reference to thefollowing appended claims, along with the full scope of equivalents towhich such claims are entitled.

[0322] All patents, patent applications and publications cited in thisapplication are hereby incorporated by reference in their entirety forall purposes to the same extent as if each individual patent, patentapplication or publication were so individually denoted.

What is claimed is:
 1. A method of determining the electrical potentialacross a membrane comprising: (a) introducing a first reagent comprisinga hydrophobic fluorescent ion capable of redistributing from a firstface of the membrane to a second face of the membrane in response tochanges in the membrane potential; (b) introducing a second reagentwhich labels the first face or the second face of the membrane, whichsecond reagent comprises a chromophore capable of undergoing energytransfer by either (i) donating excited state energy to the fluorescention, or (ii) accepting excited state energy from the fluorescent ion;(c) exposing the membrane to excitation light; (d) measuring energytransfer between the fluoresent ion and the second reagent; and (e)relating the energy transfer to the membrane potential.
 2. The method ofclaim 1, wherein energy transfer between the fluorescent ion and thesecond reagent is by fluorescent resonance energy transfer (FRET). 3.The method of claim 1, wherein the membrane is a plasma membrane of abiological cell.
 4. The method of claim 3, wherein the cell is amammalian cell.
 5. The method of claim 4, wherein the membrane is in anintracellular organelle.
 6. The method of claim 4, wherein the cell isselected from the group consisting of L-M (TK⁻) cells, neuroblastomacells, astrocytoma cells and neonatal cardiac myocytes.
 7. The method ofclaim 1, wherein the membrane comprises a phospholipid bilayer.
 8. Themethod of claim 1, wherein the ion is an anion.
 9. The method of claim8, wherein the anion bears a single charge.
 10. The method of claim 8,wherein the anion is selected from the group consisting of polymethineoxonols, tetraaryl borates and complexes of transition metals.
 11. Themethod of claim 10, wherein the anion is an oxonol of the formula

wherein: R is independently selected from the group consisting of H,hydrocarbyl and heteroalkyl; X is oxygen or sulfur; and n is an integerfrom 1 to 3; and salts thereof.
 12. The method of claim 11, wherein X issulfur.
 13. The method of claim 11, wherein: each R is identical and isa hydrocarbyl group selected from C₁₋₁₀ alkyl groups; and n=2.
 14. Themethod of claim 10, wherein the anion is a tetraaryl borate of theformula [(Ar¹)₃B-Ar²-Y-FLU]⁻ wherein: Ar¹ is an aryl group; Ar² is anarylene group; B is boron; Y is oxygen or sulfur; and FLU is a neutralfluorophore; and derivatives thereof.
 15. The method of claim 14,wherein: Ar¹ is trifluoromethylphenyl; Ar² is tetrafluorophenyl; and Yis oxygen.
 16. The method of claim 14, wherein the neutral fluorophoreis selected from the group consisting of bimanes,difluoroboradiazaindacenes and coumarins.
 17. The method of claim 16,wherein the neutral fluorophore is a bimane of the formula

wherein: each R⁵, which may be the same or different, is independentlyH, lower alkyl or an alkylene attachment point.
 18. The method of claim16, wherein the neutral fluorophore is a difluoroboradiazaindacene ofthe formula

wherein: each R¹, which may be the same or different, is independentlyselected from the group consisting of H, lower alkyl, aryl,heteroaromatic, aralkenyl and an alkylene attachment point; each R²,which may be the same or different, is independently selected from thegroup consisting of H, lower alkyl, phenyl and an alkylene attachmentpoint.
 19. The method of claim 16, wherein the neutral fluorophore is acoumarin of the formulas

wherein: each R³, which may be the same or different, is independentlyselected from the group consisting of H, halogen, lower alkyl, CN, CF₃and OR⁵; each R⁴, which may be the same or different, is selected fromthe group consisting of H, OR⁵ and an alkylene attachment point; and Zis O, S or NR″; and R⁵ is selected from the group consisting of H, loweralkyl and an alkylene attachment point.
 20. The method of claim 16,wherein the neutral fluorophore is a complex of a transition metal ofthe formula

wherein: Ln−Tb, Eu, or Sm; R is independently H, C1-C8 alkyl, C1-C8cycloalkyl or C1-C4 perfluoroalkyl; X and Y are independently H, F, C1,Br, I, NO₂, CF₃, lower (C1-C4) alkyl, CN, Ph, O-(lower alkyl), or OPh;or X and Y together are —CH═CH—; and Z=alkylenediyl, heteroalkylenediylor heterocyclodiyl.
 21. The method of claim 20, whereinZ=1,2-ethanediyl, 1,3-propanediyl, 2,3-butanediyl, 1,2-cyclohexanediyl,1,2-cyclopentanediyl, 1,2-cycloheptanediyl, 1,2-phenylenediyl,3-oxa-1,5-pentanediyl, 3-aza-3-(lower alkyl)-1,5-pentanediyl,pyridine-2,6-bis(methylene) or tetrahydrofuran-2,5-bis(methylene). 22.The method of claim 16, wherein the neutral fluorophore is a complex ofa transition metal of the formula

wherein: Ln=Tb, Eu, or Sm; R is independently H, C1-C8 alkyl, C1-C8cycloalkyl or C1-C4 perfluoroalkyl; X′ and Y′ are independently H, F,C1, Br, I, NO₂, CF₃, lower (C1-C4) alkyl, CN, Ph, O-(lower alkyl), orOPh; or X′ and Y′ together are —CH═CH—; and Z′ is independently avalence bond, CR₂, pyridine-2-6-diyl or tetrahydofuran-2,5-diyl.
 23. Themethod of claim 1, wherein the second reagent is a fluorophore.
 24. Themethod of claim 23, wherein the second reagent is selected from thegroup consisting of lectins, lipids, carbohydrates, cytochromes andantibodies, each being labelled with a fluorophore.
 25. The method ofclaim 24, wherein the fluorophore is selected from the group consistingof xanthenes, cyanines and coumarins.
 26. The method of claim 24,wherein the second reagent is a lipid which is a phospholipid.
 27. Themethod of claim 1, wherein the first reagent and the second reagent arecovalently joined by a linker.
 28. The method of claim 27, wherein thelinker is a compound of the formula:X-(CH₂)_(m)-Z_(q)-(CH₂)_(m′)-Z′_(q′)-(CH₂)_(m″)-Z″_(q″)-Y wherein: X isa hydrophobic fluorescent anion selected from the group consisting ofpolymethine oxonols and fluorescent tetraaryl borates; Y is afluorescent second reagent selected from the group consisting of lectinsand phospholipids; Z, Z′, Z″ are independently O, S, SS, CO, COO; m, m′and m″ are integers from 0 to about 32; q, q′, and q″ are independently0 or 1; and m+q+m′+q′+m″+q″ is from about 20 to
 40. 29. The method ofclaim 28, wherein the linker is a thioether.
 30. A kit comprising: (a) afirst reagent comprising a hydrophobic fluorescent ion capable ofredistributing from a first face of a membrane to a second face of themembrane in response to changes in the membrane potential; and (b) asecond reagent which labels the first face or the second face of themembrane, which second reagent comprises a chromophore capable ofundergoing energy transfer by either (i) donating excited state energyto the fluorescent ion, or (ii) accepting excited state energy from thefluorescent ion.
 31. The kit of claim 30, wherein: the first reagent isselected from the group consisting of polymethine oxonols, tetraarylborates and complexes of transition metals; and the second reagent isselected from the group consisting of lectins, lipids, carbohydrates,cytochromes and antibodies, each being labelled with a fluorophore. 32.The kit of claim 30, further comprising a solubilizing agent.
 33. Acompound of the formula A-L-B wherein: A is independently a polymethineoxonol or a tetraaryl borate linked to a fluorophore; L is a linker; andB is a membrane-impermeant fluorophore or a membrane-impermeantconjugate of a fluorophore.
 34. The compound of claim 33 wherein A is apolymethine oxonol of the formula:

wherein: R is independently selected from the group consisting of H,hydrocarbyl and heteroalkyl; X is oxygen or sulfur; and n is an integerfrom 1 to 3; provided that at least one R is an alkylene group.
 35. Acompound of a formula Cou-PE wherein: Cou is a coumarin of formula:

wherein: each R³, which may be the same or different, is independentlyselected from the group consisting of H, halogen, lower alkyl, CN, CF₃,COOR₅, CON(R⁵)₂, OR⁵, and an attachment point; R⁴ is selected from thegroup consisting of OR⁵ and N(R⁵)₂; Z is O, S or NR⁵; and each R⁵, whichmay be the same or different, is independently selected from the groupconsisting of H, lower alkyl and an alkylene attachment point; and PE isan N-linked phosphatidylethanolamine.
 36. A compound of a formula

wherein: Ln=Tb, Eu, or Sm; R is independently H, C1-C8 alkyl, C1-C8cycloalkyl or C1-C4 perfluoroalkyl; X and Y are independently H, F, C1,Br, I, NO₂, CF₃, lower (C1-C4) alkyl, CN, Ph, O-(lower alkyl), or OPh;or X and Y together are —CH═CH—; and Z=alkylenediyl, heteroalkylenediylor heterocyclodiyl.
 37. The compound of claim 36, wherein:Z=1,2-ethanediyl, 1,3-propanediyl, 2,3-butanediyl, 1,2-cyclohexanediyl,1,2-cyclopentanediyl, 1,2-cycloheptanediyl, 1,2-phenylenediyl,3-oxa-1,5-pentanediyl, 3-aza-3-(lower alkyl)-1,5-pentanediyl,pyridine-2,6-bis(methylene) or tetrahydrofuran-2,5-bis(methylene).
 38. Acompound of a formula

wherein: Ln=Tb, Eu, or Sm; R is independently H, C1-C8 alkyl, C1-C8cycloalkyl or C1-C4 perfluoroalkyl; X′ and Y′ are independently H, F,C1, Br, I, NO₂, CF₃, lower (C1-C4) alkyl, CN, Ph, O-(lower alkyl), orOPh; or X′ and Y′ together are —CH═CH—; and Z′ is independently avalence bond, CR₂, pyridine-2-6-diyl or tetrahydofuran-2,5-diyl.
 39. Apolymethine oxonol of the formula

wherein: X is oxygen or sulfur; R is a C₄₋₁₂ hydrocarbyl; n is aninteger from 1 to 3; provided that when n is 1, R is a C₇₋₁₂hydrocarbyl; and salts thereof.
 40. A compound of the formula:

wherein PE represents an N-linked phosphatidylethanolamine.
 41. A methodof identifying a test sample which affects membrane potential in a cell,comprising: (a) loading the cells with a first and second reagents,which together determine the membrane potential by the method of claim1; (b) exposing the membrane to the test sample; (c) determining thepotential of the membrane; and (d) comparing the potential in (c) to thepotential in the absence of the test sample, thereby determining theeffect of the test sample on the membrane potential.
 42. The method ofclaim 41, further comprising: (e) exposing the membrane to a stimuluswhich modulates the activity of an ion channel, pump or exchanger; (f)determining the membrane potential; (g) redetermining the membranepotential in the presence of the test sample; and (h) comparing themembrane potentials in (f) and (g) to determine the effect of the testsample on the stimulus.
 43. The methods of claims 41 or 42, wherein thecell is a mammalian cell.
 44. A method of screening test samples toidentify a compound which modulates the activity of an ion channel, pumpor exchanger in a membrane, comprising: (a) loading a first set and asecond set of cells with first and second reagents which togethermeasure membrane potential by the method of claim 1; (b) optionally,exposing both the first and second set of cells to a stimulus whichmodulates an ion channel, pump or exchanger; (c) exposing the first setof cells to a test sample; (d) measuring the membrane potential of thefirst and second sets of cells; and (e) relating the difference inmembrane potentials between the first and second sets of cells to theability of a compound in the test sample to modulate the activity of anion channel, pump or exchanger in the membrane.
 45. A method ofsynthesizing a fluorescent tetraryl borate of formula[(Ar¹)₃B—Ar²—Y—FLU]⁻ wherein: Ar¹ is an aryl group; Ar² is an arylenegroup; B is boron; Y is oxygen or sulfur; and FLU is a neutralfluorophore; said method comprising: (a) treating a triaryl borane(Ar¹)₃B with an organometallic reagent of formula (P—Y—Ar²)-M, wherein Pis a protecting group and M is a metal to form a protected tetraarylborate (Ar¹)₃B—Ar²—Y—P; (b) removing the protecting group from theprotected tetraaryl borate to form (Ar¹)₃B—Ar²—YH; and (c) treating(Ar¹)₃B—Ar²—YH with a fluorophore FLU bearing a leaving group to form afluorescent tetraryl borate.
 46. A compound of the formula

wherein: each R′ is independently H, hydrocarbyl, halogen, CF₃ or alinker group; n is an integer from 0 to 5; each X is independently H,halogen or CF₃; m is an integer from 0 to 4; Y is oxygen or sulfur; andFLU is a neutral fluorophore selected from the group consisting ofxanthenes, coumarins, cyanines, bimanes and difluoroboradiazaindacenes.